Novel Beta-Galactoside Alpha 2,6-Sialyltransferase, Gene Coding For The Transferase And Process For Producing The Same

ABSTRACT

The present invention provides a novel β-galactoside-α2,6-sialyltransferase having high productivity and/or high activity, and a nucleic acid encoding the sialyltransferase. The present invention also provides a microorganism producing the sialyltransferase. The present invention further provides a vector carrying a nucleic acid encoding the sialyltransferase, and a host cell transformed with the vector, as well as a method for producing a recombinant β-galactoside-α2,6-sialyltransferase.

TECHNICAL FIELD

The present invention relates to a novelβ-galactoside-α2,6-sialyltransferase, a gene encoding the enzyme, amicroorganism producing the enzyme and a method for producing theenzyme.

BACKGROUND ART

Glycosyltransferases are enzymes involved in in vivo biosynthesis ofsugar chains on glycoproteins, glycolipids and the like (hereinafterreferred to as “complex carbohydrates”). Their reaction products, i.e.,sugar chains on complex carbohydrates have very important functions inthe body. For example, sugar chains have been shown to be importantmolecules primarily in mammalian cells, which play a role in cell-celland cell-extracellular matrix signaling and serve as tags for complexcarbohydrates during differentiation and/or development.

Erythropoietin, a hormone for blood erythrocyte production, can bepresented as an example where sugar chains are applied.Naturally-occurring erythropoietin is disadvantageous in that it has ashort-lasting effect. Although erythropoietin is inherently aglycoprotein, further attempts have been made to add new sugar chainsonto erythropoietin, as a result of which recombinant erythropoietinproteins with an extended in vivo life span have been developed andproduced and are now commercially available. In the future, there willbe increasing development of such products in which sugar chains areadded or modified, including pharmaceuticals and functional foods. Thus,it is required to develop a means for freely synthesizing and producingsugar chains. In particular, the development of glycosyltransferases isincreasing in importance as one of the most efficient means.

Until now, about 150 or more glycosyltransferase genes have beenisolated from eukaryotic organisms including humans, mice, rats andyeast. Moreover, these genes have been expressed in host cells such asCHO cells or E. coli cells to produce proteins havingglycosyltransferase activity. On the other hand, about 20 to 30 types ofglycosyltransferase genes have also been isolated from bacteria whichare prokaryotic organisms. Moreover, proteins having glycosyltransferaseactivity have been expressed in recombinant production systems using E.coli and identified for their substrate specificity and/or variousenzymatic properties.

Sialic acid is often located at the nonreducing termini of sugar chainsand is therefore regarded as a very important sugar in terms of allowingsugar chains to exert their functions. For this reason,sialyltransferase is one of the most in demand enzymes amongglycosyltransferases. As to β-galactoside-α2,6-sialyltransferases andtheir genes, many reports have been issued for those derived fromanimals, particularly mammals (Hamamoto, T., et al., Bioorg. Med. Chem.,1, 141-145 (1993); Weinstein, J., et al., J. Biol. Chem., 262,17735-17743 (1987)). However, such animal-derived enzymes are veryexpensive because they are difficult to purify and hence cannot beobtained in large amounts. Moreover, such enzymes have a problem in thatthey have poor stability as enzymes. In contrast, as to bacterialβ-galactoside-α2,6-sialyltransferases and their genes, reports have beenissued only for those isolated from microorganisms belonging toPhotobacterium damselae (International Publication No. WO98/38315; U.S.Pat. No. 6,255,094).

However, Photobacterium damselae-derivedβ-galactoside-α2,6-sialyltransferase has a productivity of 550 U/L whenproduced from Photobacterium damselae (Yamamoto, T., et al., Biosci.Biotechnol. Biochem., 62(2), 210-214 (1998)), while the productivity is224.5 U/L when this β-galactoside-α2,6-sialyltransferase is producedfrom E. coli cells transformed with plasmid pEBSTA178 carrying its gene(Yamamoto, T., et al., J. Biochem., 123, 94-100 (1998)). Thus, there isa demand for an enzyme having higher productivity. On the other hand,Photobacterium damselae-derived β-galactoside-α2,6-sialyltransferase hasa specific activity of 5.5 U/mg (Yamamoto, T., et al., J. Biochem., 120,104-110 (1996)). In this regard, there is also a demand for an enzymehaving higher activity.

Among known bacterial sialyltransferases, Pasteurella multocida-derivedα2,3-sialyltransferase can be listed as an enzyme whose productivity andactivity are relatively high, although it is categorized as a differenttype of enzyme. This enzyme has a productivity of 6,000 U/L (Yu, H., etal., J. Am. Chem. Soc., 127, 17618-17619 (2005)) and a specific activityof 60 U/mg.

To meet the high demand of sialyltransferases, there is a need forβ-galactoside-α2,6-sialyltransferases having higher productivity and/oractivity.

Patent Document 1: International Publication No. WO98/38315

Patent Document 2: U.S. Pat. No. 6,255,094

Non-patent Document 1: Hamamoto, T., et al., Bioorg. Med. Chem., 1,141-145 (1993)

Non-patent Document 2: Weinstein, J., et al., J. Biol. Chem., 262,17735-17743 (1987)

Non-patent Document 3: Yamamoto, T., et al., Biosci. Biotechnol.Biochem., 62(2), 210-214 (1998)

Non-patent Document 4: Yamamoto, T., et al., J. Biochem., 123, 94-100(1998)

Non-patent Document 5: Yamamoto, T., et al., J. Biochem., 120, 104-110(1996)

Non-patent Document 6: Yu, H., et al., J. Am. Chem. Soc., 127,17618-17619 (2005)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

A problem to be solved by the present invention is to provide a novelβ-galactoside-α2,6-sialyltransferase derived from a microorganismbelonging to the genus Photobacterium of the family Vibrionaceae, and agene encoding the same. The present invention also aims to provide anovel β-galactoside-α2,6-sialyltransferase having higher productivityand/or higher activity than known bacterial sialyltransferases, and agene encoding the same.

Another problem to be solved by the present invention is to provide amethod for high production of the β-galactoside-α2,6-sialyltransferaseof the present invention by gene recombination technology using a geneencoding this enzyme.

Means for Solving the Problems

As a result of extensive and intensive efforts made to separate andcharacterize 4,000 or more microbial strains from all areas of Japan,the inventors of the present invention have found a strain producingβ-galactoside-α2,6-sialyltransferase activity from among strains ofmicroorganisms belonging to the genus Photobacterium. The inventors havethen cloned a novel α2,6-sialyltransferase gene from this strain byusing as a probe the DNA of a known β-galactoside-α2,6-sialyltransferasegene from Photobacterium damselae. As a result of expressing this novelgene in E. coli cells, the inventors have found that this gene encodes aprotein having β-galactoside-α2,6-sialyltransferase activity, and thatthe productivity of this enzyme is as high as about 10,700 U per literof culture solution. As a result of further efforts to purify andanalyze in detail this novel recombinant enzyme, the inventors have alsofound that this recombinant enzyme efficiently transfers sialic acid inα2,6 linkage to galactose, N-acetylgalactosamine or other residues insugar chains, and that its specific activity is as high as about 110 U(unit)/mg to about 260 U/mg. In this way, the inventors havedemonstrated many advantages over the knownβ-galactoside-α2,6-sialyltransferase derived from Photobacteriumdamselae, thereby completing the present invention. The presentinvention provides a novel β-galactoside-α2,6-sialyltransferase havinghigh productivity and/or high activity, and a nucleic acid encoding thesame, as well as a method for producing the sialyltransferase.

The present invention will now be illustrated in detail below.

β-Galactoside-α2,6-Sialyltransferase

The present invention provides a novelβ-galactoside-α2,6-sialyltransferase. As used herein, the term“β-galactoside-α2,6-sialyltransferase” is intended to mean a proteinhaving the ability to transfer sialic acid from cytidine monophosphate(CMP)-sialic acid to the 6-position of a galactose residue in complexcarbohydrate sugar chains or free sugar chains, to the 6-position ofgalactose present in oligosaccharides such as lactose orN-acetyllactosamine, or to the 6-position of a monosaccharide (e.g.,galactose, N-acetylgalactosamine, glucose, N-acetylglucosamine ormannose) which may be used as a constituting member of complexcarbohydrates and has a hydroxyl group on the carbon at the 6-position.As used herein, the term “β-galactoside-α2,6-sialyltransferase activity”is intended to mean the ability described above forβ-galactoside-α2,6-sialyltransferase. The term “sialic acid” as usedherein refers to a neuraminic acid derivative belonging to the sialicacid family. More specifically, it refers to N-acetylneuraminic acid(Neu5Ac), N-glycolylneuraminic acid (Neu5Gc),5-deamino-5-hydroxyneuraminic acid (KDN), disialic acid (i.e.,di-N-acetylneuraminic acid; Neu5Acα2,8(9)Neu5Ac) or the like.

The β-galactoside-α2,6-sialyltransferase of the present invention is aprotein comprising the amino acid sequence shown in SEQ ID NO: 2, SEQ IDNO: 4 or SEQ ID NO: 12. The amino acid sequence shown in SEQ ID NO: 4corresponds to a sequence having methionine at the N-terminus of anamino acid sequence covering amino acids 18-514 of SEQ ID NO: 2. Theamino acid sequence shown in SEQ ID NO: 12 corresponds to a sequencehaving methionine at the N-terminus of an amino acid sequence coveringamino acids 111-514 of SEQ ID NO: 2. This N-terminal methionine isderived from the initiation codon for protein expression and does notaffect the activity of β-galactoside-α2,6-sialyltransferase. Moreover,the N-terminal methionine in a protein may often be cleaved off byintracellular processing. Thus, a protein comprising the amino acidsequence shown in SEQ ID NO: 4 includes not only a protein comprising anamino acid sequence completely identical with SEQ ID NO: 4, but also aprotein comprising an amino acid sequence lacking the N-terminalmethionine.

As described later in Example 2, ISH224-N1C0/pTrc (SEQ ID NO: 4; asequence having methionine at the N-terminus of an amino acid sequencecovering amino acids 18-514 of SEQ ID NO: 2) and ISH224-N3C0/pTrc (SEQID NO: 12; a sequence having methionine at the N-terminus of an aminoacid sequence covering amino acids 111-514 of SEQ ID NO: 2) bothretained the activity of β-galactoside-α2,6-sialyltransferase derivedfrom the strain JT-ISH-224. Thus, the presence of at least amino acids111-514 of SEQ ID NO: 2 allows retention ofβ-galactoside-α2,6-sialyltransferase activity. For this reason, the“protein comprising the amino acid sequence shown in SEQ ID NO: 12”according to the present invention includes a protein comprising anamino acid sequence lacking all or part of amino acids 1-110 from aminoacids 1-514 of SEQ ID NO: 2.

Alternatively, the β-galactoside-α2,6-sialyltransferase of the presentinvention is a protein encoded by a nucleic acid comprising thenucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:11. The nucleotide sequence shown in SEQ ID NO: 3 corresponds to asequence having an initiation codon (ATG) at the 5′-terminus of anucleotide sequence covering nucleotides 52-1545 of SEQ ID NO: 1. Thenucleotide sequence shown in SEQ ID NO: 11 corresponds to a sequencehaving an initiation codon (ATG) at the 5′-terminus of a nucleotidesequence covering nucleotides 331-1545 of SEQ ID NO: 1. The nucleotidesequences shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 11 encodethe amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO:12, respectively.

In the β-galactoside-α2,6-sialyltransferase of the present inventioncomprising the amino acid sequence shown in SEQ ID NO: 2, a sequencecovering amino acids 12-15 of SEQ ID NO: 2 is Leu-Thr-Ala-Cys, which isa consensus sequence called lipobox, so that cleavage will occur withinbacterial cells at the amino terminus of Cys in this consensus sequence(Madan Babu, M. and Sankaran, K. Bioinformatics. 18, 641-643 (2002)).Thus, the β-galactoside-α2,6-sialyltransferase of the present inventionmay be a protein comprising an amino acid sequence covering amino acids15-514 of SEQ ID NO: 2. Alternatively, theβ-galactoside-α2,6-sialyltransferase of the present invention may be aprotein encoded by a nucleic acid comprising a nucleotide sequencecovering nucleotides 43-1545 of SEQ ID NO: 1.

The present invention also encompasses mutants of the aboveβ-galactoside-α2,6-sialyltransferases of the present invention, i.e.,mutated proteins having β-galactoside-α2,6-sialyltransferase activity.Such mutated proteins also fall within the scope of theβ-galactoside-α2,6-sialyltransferase of the present invention.

The mutant protein of the present invention may be a protein havingβ-galactoside-α2,6-sialyltransferase activity, which comprises an aminoacid sequence comprising deletion, substitution, insertion and/oraddition of one or more amino acids in an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, amino acids 15-514 of SEQ IDNO: 2, SEQ ID NO: 4 and SEQ ID NO: 12. The substitution may beconservative, which means the replacement of a certain amino acidresidue by another residue having similar physical and chemicalcharacteristics. Non-limiting examples of conservative substitutioninclude replacement between aliphatic group-containing amino acidresidues such as Ile, Val, Leu or Ala, and replacement between polarresidues such as Lys-Arg, Glu-Asp or Gln-Asn replacement.

Mutants derived by amino acid deletion, substitution, insertion and/oraddition can be prepared when DNAs encoding their wild-type proteins aresubjected to, for example, well-known site-directed mutagenesis (see,e.g., Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, whichis hereby incorporated by reference in its entirety). As used herein,the term “one or more amino acids” is intended to mean a possible numberof amino acids which may be deleted, substituted, inserted and/or addedby site-directed mutagenesis.

Site-directed mutagenesis may be accomplished, for example, as followsusing a synthetic oligonucleotide primer that is complementary tosingle-stranded phage DNA to be mutated, except for having a specificmismatch (i.e., a desired mutation). Namely, the above syntheticoligonucleotide is used as a primer to cause synthesis of acomplementary strand by phages, and the resulting duplex DNA is thenused to transform host cells. The transformed bacterial culture isplated on agar, whereby plaques are allowed to form fromphage-containing single cells. As a result, in theory, 50% of newcolonies contain phages with the mutation as a single strand, while theremaining 50% have the original sequence. At a temperature which allowshybridization with DNA completely identical to one having the abovedesired mutation, but not with DNA having the original strand, theresulting plaques are allowed to hybridize with a synthetic probelabeled by kinase treatment. Subsequently, plaques hybridized with theprobe are picked up and cultured for collection of their DNA.

Techniques for allowing deletion, substitution, insertion and/oraddition of one or more amino acids in the amino acid sequences ofbiologically active peptides such as enzymes while retaining theiractivity include site-directed mutagenesis mentioned above, as well asother techniques such as those for treating a gene with a mutagen, andthose in which a gene is selectively cleaved to remove, substitute,insert or add a selected nucleotide or nucleotides, and then ligated.

The mutant protein of the present invention may also be a protein havingβ-galactoside-α2,6-sialyltransferase activity, which is encoded by anucleic acid comprising a nucleotide sequence comprising deletion,substitution, insertion and/or addition of one or more nucleotides in anucleotide sequence selected from the group consisting of SEQ ID NO: 1,nucleotides 43-1545 of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 11.Nucleotide deletion, substitution, insertion and/or addition may beaccomplished by site-directed mutagenesis or other techniques asmentioned above.

The mutant protein of the present invention may further be a proteinhaving β-galactoside-α2,6-sialyltransferase activity, which comprises anamino acid sequence sharing an amino acid identity of at least 60% ormore, preferably 65% or more, 70% or more, 75% or more, 80% or more, 85%or more, 90% or more, 95% or more, 98% or more, or 99% or more, and morepreferably 99.5% or more with an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, amino acids 15-514 of SEQ ID NO: 2,SEQ ID NO: 4 and SEQ ID NO: 12.

Alternatively, the mutant protein of the present invention may be aprotein having β-galactoside-α2,6-sialyltransferase activity, which isencoded by a nucleic acid sharing an identity of at least 70% or more,preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 98% or more, or 99% or more, and more preferably 99.5% or morewith a nucleotide sequence selected from the group consisting of SEQ IDNO: 1, nucleotides 43-1545 of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO:11.

The percent identity between two amino acids may be determined by visualinspection and mathematical calculation. Alternatively, the percentidentity of two protein sequences may be determined by comparingsequence information based on the algorithm of Needleman, S. B. andWunsch, C. D. (J. Mol. Biol., 48:443-453, 1970) and using the GAPcomputer program available from the University of Wisconsin GeneticsComputer Group (UWGCG). The preferred default parameters for the GAPprogram include: (1) a scoring matrix, blosum62, as described byHenikoff, S. and Henikoff, J. G. (Proc. Natl. Acad. Sci. USA,89:10915-10919, 1992); (2) a gap weight of 12; (3) a gap length weightof 4; and (4) no penalty for end gaps.

Other programs used by those skilled in the art of sequence comparisonmay also be used. The percent identity can be determined by comparingsequence information using, e.g., the BLAST program described byAltschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997). Thisprogram is available on the Internet at the web site of the NationalCenter for Biotechnology Information (NCBI) or the DNA Data Bank ofJapan (DDBJ). The details of various conditions (parameters) foridentity search using the BLAST program are shown on these web sites,and default values are commonly used for search although part of thesettings may be changed as appropriate. Alternatively, the percentidentity of two amino acid sequences may be determined by using aprogram such as genetic information processing software GENETYX Ver. 7(Genetyx Corporation, Japan) or using an algorithm such as FASTA. Inthis case, default values may be used for search.

The percent identity between two nucleic acid sequences can bedetermined by visual inspection and mathematical calculation, or morepreferably, the comparison is done by comparing sequence informationusing a computer program. An exemplary, preferred computer program isthe Genetic Computer Group (GCG; Madison, Wis.) Wisconsin packageversion 10.0 program, “GAP” (Devereux et al., 1984, Nucl. Acids Res.,12:387). In addition to making a comparison between two nucleic acidsequences, this “GAP” program can be used for comparison between twoamino acid sequences and between a nucleic acid sequence and an aminoacid sequence. The preferred default parameters for the “GAP” programinclude: (1) the GCG implementation of a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted amino acid comparison matrix of Gribskovand Burgess, Nucl. Acids Res., 14:6745, 1986, as described by Schwartzand Dayhoff, eds., “Atlas of Polypeptide Sequence and Structure,”National Biomedical Research Foundation, pp. 353-358, 1979, or othercomparable comparison matrices; (2) a penalty of 30 for each gap and anadditional penalty of 1 for each symbol in each gap for amino acidsequences, or penalty of 50 for each gap and an additional penalty of 3for each symbol in each gap for nucleotide sequences; (3) no penalty forend gaps; and (4) no maximum penalty for long gaps. Other programs usedby those skilled in the art of sequence comparison can also be used,such as, for example, the BLASTN program version 2.2.7, available foruse via the National Library of Medicine website:http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.html, or the UW-BLAST 2.0algorithm. Standard default parameter settings for UW-BLAST 2.0 aredescribed at the following Internet site: http://blast.wustl.edu. Inaddition, the BLAST algorithm uses the BLOSUM62 amino acid scoringmatrix, and optional parameters that can be used are as follows: (A)inclusion of a filter to mask segments of the query sequence that havelow compositional complexity (as determined by the SEG program ofWootton and Federhen (Computers and Chemistry, 1993); also see Woottonand Federhen, 1996, “Analysis of compositionally biased regions insequence databases,” Methods Enzymol., 266: 554-71) or segmentsconsisting of short-periodicity internal repeats (as determined by theXNU program of Clayerie and States (Computers and Chemistry, 1993)), and(B) a statistical significance threshold for reporting matches againstdatabase sequences, or E-score (the expected probability of matchesbeing found merely by chance, according to the stochastic model ofKarlin and Altschul, 1990; if the statistical significance ascribed to amatch is greater than this E-score threshold, the match will not bereported.); preferred E-score threshold values are 0.5, or in order ofincreasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, le-5,le-10, le-15, le-20, le-25, le-30, le-40, le-50, le-75, or le-100.

The mutant protein of the present invention may also be a protein havingβ-galactoside-α2,6-sialyltransferase activity, which is encoded by anucleic acid comprising a nucleotide sequence hybridizable understringent conditions with the complementary strand of a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, nucleotides43-1545 of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 11.

The term “under stringent condition” means that two sequences hybridizeunder moderately or highly stringent conditions. More specifically,moderately stringent conditions can be readily determined by thosehaving ordinary skill in the art, e.g., depending on the length of DNA.The basic conditions are set forth by Sambrook et al., MolecularCloning: A Laboratory Manual, third edition, chapters 6 and 7, ColdSpring Harbor Laboratory Press, 2001 and include the use of a prewashingsolution for nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0), hybridization conditions of about 50% formamide, 2×SSC to 6×SSC atabout 40-50° C. (or other similar hybridization solutions, such asStark's solution, in about 50% formamide at about 42° C.) and washingconditions of, for example, about 40-60° C., 0.5-6×SSC, 0.1% SDS.Preferably, moderately stringent conditions include hybridization (andwashing) at about 50° C. and 6×SSC. Highly stringent conditions can alsobe readily determined by those skilled in the art, e.g., depending onthe length of DNA.

Generally, such conditions include hybridization and/or washing athigher temperature and/or lower salt concentration (such ashybridization at about 65° C., 6×SCC to 0.2×SSC, preferably 6×SCC, morepreferably 2×SSC, most preferably 0.2×SSC), compared to the moderatelystringent conditions. For example, highly stringent conditions mayinclude hybridization as defined above, and washing at approximately65-68° C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15 M NaCl, 10 mMNaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is0.15 M NaCl and 15 mM sodium citrate) in the hybridization and washingbuffers; washing is performed for 15 minutes after hybridization iscompleted.

It is also possible to use a commercially available hybridization kitwhich uses no radioactive substance as a probe. Specific examplesinclude hybridization with an ECL direct labeling & detection system(Amersham). Stringent conditions include, for example, hybridization at42° C. for 4 hours using the hybridization buffer included in the kit,which is supplemented with 5% (w/v) Blocking reagent and 0.5 M NaCl, andwashing twice in 0.4% SDS, 0.5×SSC at 55° C. for 20 minutes and once in2×SSC at room temperature for 5 minutes.

Sialyltransferase activity may be measured by known procedures, e.g.,those described in J. Biochem., 120, 104-110 (1996) (which is herebyincorporated by reference in its entirety). For example, the enzymeactivity can be evaluated by effecting an enzymatic reaction usingCMP-NeuAc (N-acetylneuraminic acid) as a glycosyl donor substrate andlactose as a glycosyl acceptor substrate, followed by evaluating theamount of the reaction product sialyllactose. It should be noted thatone enzyme unit (1 U) is defined as the amount of enzyme required totransfer 1 micromole of sialic acid per minute.

Determination of the binding mode of sialic acid transferred to aglycosyl acceptor substrate may be accomplished by using, but notlimited to, any procedure known to those skilled in the art, such asthose using a pyridylaminated sugar chain or reaction product analysisby nuclear magnetic resonance spectroscopy (NMR). Procedures using apyridylaminated sugar chain comprise effecting an enzymatic reactionusing a pyridylaminated sugar chain as a glycosyl acceptor substrate.More specifically, an enzymatic reaction is effected usingpyridylaminated lactose (Galβ1-4Glc-PA, Takara Bio Inc., Japan) as aglycosyl acceptor substrate and CMP-NeuAc as a glycosyl donor substrate,and the reaction product is subjected to high performance liquidchromatography (HPLC) analysis. From the retention time of the reactionproduct, the position at which sialic acid was transferred isidentified.

In an embodiment of the present invention, the enzyme of the presentinvention is derived from microorganisms belonging to the genusPhotobacterium. The enzyme of the present invention is not limited inany way as long as it is derived from microorganisms belonging to thegenus Photobacterium. It may be an enzyme derived from a new species ofmicroorganism belonging to the genus Photobacterium.

As to enzymological properties as well as physical and chemicalproperties, the β-galactoside-α2,6-sialyltransferase of the presentinvention is not only characterized by havingβ-galactoside-α2,6-sialyltransferase activity as defined above, but alsohas additional properties including, but not limited to: an optimum pHranging from 5 to 6; an optimum temperature of 25° C. to 35° C.; and amolecular weight of about 56,000±3,000 Da, as measured by SDS-PAGEanalysis.

Moreover, in an embodiment, the β-galactoside-α2,6-sialyltransferase ofthe present invention is characterized by having highβ-galactoside-α2,6-sialyltransferase activity. As used herein, the term“high β-galactoside-α2,6-sialyltransferase activity” is intended to meanhaving activity of 6 U or more, 10 U or more, 20 U or more, 40 U ormore, 60 U or more, or 100 U or more per mg of enzyme.

Nucleic Acid Encoding β-Galactoside-α2,6-Sialyltransferase

The present invention provides a nucleic acid encodingβ-galactoside-α2,6-sialyltransferase.

The nucleic acid of the present invention is a nucleic acid encoding aprotein comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, amino acids 15-514 of SEQ ID NO: 2, SEQ IDNO: 4 and SEQ ID NO: 12. Alternatively, the nucleic acid of the presentinvention is a nucleic acid comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, nucleotides 43-1545 of SEQ IDNO: 1, SEQ ID NO: 3 and SEQ ID NO: 11.

The nucleic acid of the present invention may be a mutant of the abovenucleic acid as long as it is a nucleic acid encoding a protein havingβ-galactoside-α2,6-sialyltransferase activity. Such a nucleic acid alsofalls within the scope of the nucleic acid of the present inventionencoding β-galactoside-α2,6-sialyltransferase.

Such a nucleic acid mutant is a nucleic acid encoding a protein havingβ-galactoside-α2,6-sialyltransferase activity, wherein the proteincomprises an amino acid sequence comprising deletion, substitution,insertion and/or addition of one or more amino acids in an amino acidsequence selected from the group consisting of SEQ ID NO: 2, amino acids15-514 of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 12. The nucleic acidmutant of the present invention is also a nucleic acid comprising anucleotide sequence comprising deletion, substitution, insertion and/oraddition of one or more nucleotides in a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, nucleotides 43-1545 of SEQ IDNO: 1, SEQ ID NO: 3 and SEQ ID NO: 11. Amino acid or nucleotidedeletion, substitution, insertion and/or addition can be introduced asdescribed above.

Alternatively, such a nucleic acid mutant is a nucleic acid encoding aprotein having β-galactoside-α2,6-sialyltransferase activity, whereinthe protein comprises an amino acid sequence sharing an identity of atleast 60% or more, preferably 65% or more, 70% or more, 75% or more, 80%or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% ormore, and more preferably 99.5% or more with an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, amino acids 15-514of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 12. The nucleic acid mutantof the present invention is also a nucleic acid encoding a proteinhaving β-galactoside-α2,6-sialyltransferase activity, wherein thenucleic acid shares an identity of preferably 70% or more, 75% or more,80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99%or more, and more preferably 99.5% or more with a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, nucleotides 43-1545of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 11. In this case, theidentity between amino acid sequences or nucleotide sequences can bedetermined as described above.

Such a nucleic acid mutant is further a nucleic acid encoding a proteinhaving β-galactoside-α2,6-sialyltransferase activity, wherein thenucleic acid comprises a nucleotide sequence hybridizable understringent conditions or highly stringent conditions with thecomplementary strand of a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, nucleotides 43-1545 of SEQ ID NO: 1, SEQ IDNO: 3 and SEQ ID NO: 11. In this case, stringent conditions or highlystringent conditions are as defined above.

Microorganisms Expressing β-Galactoside-α-2,6-Sialyltransferase

The inventors of the present invention have found that microorganismsbelonging to the genus Photobacterium of the family Vibrionaceae expressa novel β-galactoside-α2,6-sialyltransferase. Thus, the presentinvention provides microorganisms expressingβ-galactoside-α2,6-sialyltransferase. The microorganisms of the presentinvention are those belonging to the genus Photobacterium and having theability to produce β-galactoside-α2,6-sialyltransferase. Examples ofmicroorganisms belonging to the genus Photobacterium and having theability to produce β-galactoside-α2,6-sialyltransferase includePhotobacterium sp. strain JT-ISH-224 (Accession No. NITE BP-87). Itshould be noted that the above microorganisms of the genusPhotobacterium are generally marine bacteria, which are separated fromsea water or marine products such as fish and shellfish. For example,Photobacterium sp. strain JT-ISH-224 of the present invention wasseparated from barracuda in Ishikawa prefecture.

The microorganisms of the present invention can be separated usingscreening procedures as shown below, by way of example. Sea water, seasand, sea mud or a marine product is used as a microorganism source. Seawater, sea sand and sea mud may be used directly or further diluted withsterilized sea water for use as an inoculum. In the case of small marineanimals, their surface slime or the like is collected by scrubbing witha loop and is then used as an inoculum; or alternatively, their internalorgans are homogenized in sterilized sea water and the resulting fluidis used as an inoculum. These inocula are applied onto agar plates suchas marine broth agar 2216 medium (Becton Dickinson) or sodiumchloride-supplemented nutrient agar medium (Becton Dickinson) to obtainmarine microorganisms growing under various temperature conditions.After the resulting microorganisms have been pure-cultured in a routinemanner, each microorganism is cultured using a liquid medium such asmarine broth 2216 medium (Becton Dickinson) or sodiumchloride-supplemented nutrient broth medium (Becton Dickinson). Afterthe microorganisms are fully grown, the cells are collected bycentrifugation from each culture solution. To the collected cells, 20 mMcacodylate buffer (pH 6.0) containing 0.2% Triton X-100 (Kanto Kagaku,Japan) is added, and the cells are suspended therein. This cellsuspension is ultrasonicated under ice cooling to homogenize the cells.This cell homogenate is used as an enzyme solution and measured for itssialyltransferase activity in a routine manner, to thereby obtain astrain having sialyltransferase activity.

The above screening procedures were also used for obtainingPhotobacterium sp. strain JT-ISH-224 of the present invention. Itsmicrobiological properties as well as physiological and biochemicalproperties will be detailed in Example 1, along with speciesidentification based on nucleotide sequence analysis of the 16S-rRNAgene.

Photobacterium sp. strain JT-ISH-224 was deposited under the BudapestTreaty on Mar. 11, 2005 under NITE BP-87 with the National Institute ofTechnology and Evaluation, Patent Microorganisms Depositary (NPMD; 2-5-8Kazusakamatari, Kisarazu, Chiba, Japan).

Method for Producing β-Galactoside-α2,6-Sialyltransferase

The present invention also relates to a method for producing theβ-galactoside-α2,6-sialyltransferase of the present invention. In apreferred embodiment, the method of the present invention allows highproduction of the enzyme of the present invention. More specifically,the productivity of the enzyme of the present invention in the method ofthe present invention is 50 U/L or more, 1,000 U/L or more, or 10,000U/L or more per liter of culture solution.

(1) Method for Producing β-galactoside-α2,6-sialyltransferase byCulturing Microorganisms Expressing the Enzyme

In an embodiment of the present invention, theβ-galactoside-α2,6-sialyltransferase of the present invention is derivedfrom microorganisms belonging to the genus Photobacterium, and isobtained as follows: a microorganism having the ability to produceβ-galactoside-α2,6-sialyltransferase is cultured in a medium and allowedto produce β-galactoside-α2,6-sialyltransferase, which is thencollected.

Microorganisms used for this purpose are not limited in any way as longas they belong to the genus Photobacterium and have the ability toproduce β-galactoside-α2,6-sialyltransferase. Preferred are thosebelonging to Photobacterium spp. Examples of microorganisms for use inthe method of the present invention include Photobacterium sp. strainJT-ISH-224 (Accession No. NITE BP-87).

For use in culturing the above microorganisms, the culture mediumcontains ingredients available to these microorganisms, including acarbon source, a nitrogen source and minerals. Such a carbon sourceincludes peptone, tryptone, casein lysate, meat extract and glucose,with peptone being preferred for use. As a nitrogen source, yeastextract is preferred for use. Salts include sodium chloride, ironcitrate, magnesium chloride, sodium sulfate, calcium chloride, potassiumchloride, sodium carbonate, sodium bicarbonate, potassium bromide,strontium chloride, sodium borate, sodium silicate, sodium fluoride,ammonium nitrate and disodium hydrogen phosphate, which are preferablyused in combination as appropriate.

Alternatively, marine broth 2216 medium (Becton Dickinson) containingthe above ingredients may be used. Further, artificial sea watercontaining the above salts in appropriate amounts may also be used,supplemented with peptone, yeast extract or the like. Culture conditionswill somewhat vary depending on the medium composition and/or the typeof strain. For example, in the case of culturing Photobacterium sp.strain JT-ISH-224, the culture temperature is about 20° C. to 30° C.,preferably about 25° C. to 30° C., and the culture period is about 6 to48 hours, preferably about 15 to 24 hours.

Since a target enzyme exists within cells, any of known cellhomogenization techniques such as ultrasonic disruption, French presshomogenization, glass bead homogenization or Dynomil homogenization canbe performed to separate and purify the target enzyme from the resultingcell homogenate. In the method of the present invention, a preferredcell homogenization technique is ultrasonic disruption. For example,after centrifugation to remove solid matter from the cell homogenate,the resulting cell homogenate supernatant can be purified, e.g., bycolumn chromatography on a commercially available column such as ananion exchange column, a cation exchange column, a gel filtrationcolumn, a hydroxyapatite column, a CDP-hexanolamine agarose column, aCMP-hexanolamine agarose column and/or a hydrophobic column, as well asNative-PAGE, which are used in combination as appropriate.

It should be noted that although β-galactoside-α2,6-sialyltransferasemay be completely purified, the β-galactoside-α2,6-sialyltransferase ofthe present invention may be in either purified or partially purifiedform because it has sufficient activity even in partially purified form.

(2) Method for Producing Recombinantβ-Galactoside-α2,6-Sialyltransferase

The present invention provides an expression vector carrying a nucleicacid encoding β-galactoside-α2,6-sialyltransferase, and a host cellcontaining the expression vector. Moreover, the present invention alsoprovides a method for producing a recombinantβ-galactoside-α2,6-sialyltransferase protein, which comprises culturinga host cell containing the expression vector under conditions suitablefor recombinant protein expression, and collecting the expressedrecombinant protein.

To produce the recombinant β-galactoside-α2,6-sialyltransferase proteinof the present invention, an expression vector chosen depending on thehost to be used is inserted with a nucleic acid sequence encodingβ-galactoside-α2,6-sialyltransferase that is operably linked to asuitable transcription or translation regulatory nucleotide sequencederived from a gene of mammalian, microorganism, viral, insect or otherorigin. Examples of such a regulatory sequence include a transcriptionpromoter, an operator or an enhancer, a mRNA ribosome binding site, aswell as suitable sequences regulating the initiation and termination oftranscription and translation.

Such a nucleic acid sequence encodingβ-galactoside-α2,6-sialyltransferase to be inserted into the vector ofthe present invention is a nucleotide sequence of the above nucleic acidof the present invention encoding β-galactoside-α2,6-sialyltransferase,which may or may not comprise a leader sequence. When the nucleotidesequence comprises a leader sequence, it may be a leader sequencecorresponding to nucleotides 1-42 of SEQ ID NO: 1, or may be replaced bya leader sequence derived from other organisms. Leader sequencereplacement enables the design of an expression system which allowssecretion of the expressed protein into the extracellular environment ofhost cells.

Moreover, the recombinant β-galactoside-α2,6-sialyltransferase proteinof the present invention may also be expressed as a fusion protein byinserting a vector with a nucleic acid designed such that a nucleic acidencoding a His tag, a FLAG™ tag, glutathione-S-transferase or the likeis linked downstream of a nucleic acid encoding the enzyme. When theenzyme of the present invention is expressed as a fusion protein in thisway, such a fusion protein can facilitate purification and detection ofthe enzyme.

Host cells suitable for protein expression ofβ-galactoside-α2,6-sialyltransferase include prokaryotic cells, yeast orhigher eukaryotic cells. Suitable cloning and expression vectors for usein bacterial, fungal, yeast and mammalian host cells are described, forexample, in Pouwels et al., Cloning Vectors: A Laboratory Manual,Elsevier, N.Y., (1985) (which is hereby incorporated by reference in itsentirety).

Prokaryotic organisms include Gram-negative or Gram-positive bacteriasuch as E. coli or Bacillus subtilis. When a prokaryotic cell such as E.coli is used as a host, a β-galactoside-α2,6-sialyltransferase proteinmay be designed to have an N-terminal methionine residue for the purposeof facilitating recombinant polypeptide expression within prokaryoticcells. This N-terminal methionine may be cleaved from the expressedrecombinant α2,6-sialyltransferase protein.

Expression vectors for use in prokaryotic host cells generally containone or more phenotype selectable marker genes. Such a phenotypeselectable marker gene is, for example, a gene imparting antibioticresistance or auxotrophy. Examples of expression vectors suitable forprokaryotic host cells include commercially available plasmids such aspBR322 (ATCC37017) or derivatives thereof. pBR322 contains genes forampicillin and tetracycline resistance, and thereby facilitatesidentification of transformed cells. DNA sequences of a suitablepromoter and a nucleic acid encodingβ-galactoside-α2,6-sialyltransferase are inserted into this pBR322vector. Other commercially available vectors include, for example,pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (PromegaBiotech., Madison, Wis., United States).

Promoter sequences generally used in expression vectors for prokaryotichost cells include tac promoter, β-lactamase (penicillinase) promoter,and lactose promoter (Chang et al., Nature 275:615, 1978; and Goeddel etal., Nature 281:544, 1979, which are hereby incorporated by reference intheir entirety).

Alternatively, a recombinant β-galactoside-α2,6-sialyltransferaseprotein may be expressed in yeast host cells, preferably usingSaccharomyces (e.g., S. cerevisiae). Other genera of yeast, such asPichia or Kluyveromyces, may also be used. Yeast vectors often containan origin of replication sequence from 2μ yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. A yeast α-factor leader sequence can also beused to induce secretion of a recombinantβ-galactoside-α2,6-sialyltransferase protein. There are also known otherleader sequences that are suitable for facilitating recombinantpolypeptide secretion from yeast hosts. Procedures for yeasttransformation are described, for example, in Hinnen et al., Proc. Natl.Acad. Sci. USA, 75: 1929-1933, 1978 (which is hereby incorporated byreference in its entirety).

Mammalian or insect host cell culture systems can also be used toexpress a recombinant β-galactoside-α2,6-sialyltransferase protein.Established cell lines of mammalian origin can also be used for thispurpose. Transcription and translation control sequences for mammalianhost cell expression vectors may be obtained from the viral genome.Promoter and enhancer sequences commonly used are derived frompolyomavirus, adenovirus 2, etc. DNA sequences derived from the SV40viral genome (e.g., SV40 origin, early and late promoters, enhancers,splice sites, polyadenylation sites) may also be used to provide othergene elements for expression of structural gene sequences in mammalianhost cells. Vectors for use in mammalian host cells can be constructed,for example, by the method of Okayama and Berg (Mol. Cell. Biol., 3:280, 1983, which is hereby incorporated by reference in its entirety).

One method of the present invention for producing aβ-galactoside-α2,6-sialyltransferase protein comprises culturing hostcells transformed with an expression vector carrying a nucleic acidsequence encoding a β-galactoside-α2,6-sialyltransferase protein, underconditions allowing expression of the protein. Then, in a mannersuitable for the expression system used, theβ-galactoside-α2,6-sialyltransferase protein is collected from theculture medium or cell extract.

Means for purifying a recombinant β-galactoside-α2,6-sialyltransferaseprotein are selected, as appropriate, depending on such factors as whattype of host was used and whether the protein of the present inventionis to be secreted into the culture medium. For example, means forpurifying a recombinant β-galactoside-α2,6-sialyltransferase proteininclude column chromatography on an anion exchange column, a cationexchange column, a gel filtration column, a hydroxyapatite column, aCDP-hexanolamine agarose column, a CMP-hexanolamine agarose columnand/or a hydrophobic column, as well as Native-PAGE or combinationsthereof. Alternatively, when a recombinantβ-galactoside-α2,6-sialyltransferase is expressed in a form fused with atag or the like for easy purification, affinity chromatographictechniques may be used for purification. For example, when a histidinetag, a FLAG™ tag or glutathione-S-transferase (GST) is fused,purification can be accomplished by affinity chromatography using aNi-NTA (nitrilotriacetic acid) column, an anti-FLAG antibody-boundcolumn or a glutathione-bound column, respectively.

Although a recombinant β-galactoside-α2,6-sialyltransferase may bepurified to give an electrophoretically single band, theβ-galactoside-2,6-sialyltransferase of the present invention may be ineither purified or partially purified form because it has sufficientactivity even in partially purified form.

Antibody

The present invention provides an antibody against theβ-galactoside-α2,6-sialyltransferase protein of the present invention.The antibody of the present invention may be prepared against theβ-galactoside-α2,6-sialyltransferase protein of the present invention ora fragment thereof. A fragment of theβ-galactoside-α2,6-sialyltransferase of the present invention used forthis purpose is a fragment having a sequence comprising at least 6 aminoacids, at least 10 amino acids, at least 20 amino acids or at least 30amino acids of the amino acid sequence of the enzyme.

Such an antibody may be prepared by immunizing theβ-galactoside-α2,6-sialyltransferase of the present invention or afragment thereof into animals which are used for antibody preparation inthe art including, but not limited to, mice, rats, rabbits, guinea pigsand goats. The antibody may be either polyclonal or monoclonal. Theantibody can be prepared based on antibody preparation techniques wellknown to those skilled in the art.

The antibody of the present invention can be used for collecting theβ-galactoside-α2,6-sialyltransferase protein of the present invention byaffinity purification.

The antibody of the present invention can also be used for detecting theβ-galactoside-α2,6-sialyltransferase protein of the present invention inassays such as western blotting and ELISA.

ADVANTAGES OF THE INVENTION

By providing a novel β-galactoside-α2,6-sialyltransferase and a nucleicacid encoding the same, the present invention makes a contribution interms of providing a means for synthesizing and producing sugar chains,which are now being shown to have important functions in the body. Inparticular, the β-galactoside-α2,6-sialyltransferase of the presentinvention has higher production efficiency and higher specific activity,as well as a wider range of acceptor substrate specificity, whencompared to conventional sialyltransferases. Sialic acid is oftenlocated at the nonreducing termini of complex carbohydrate sugar chainsin the body and is a very important sugar in terms of sugar chainfunctions. Thus, sialyltransferase is one of the most in demand enzymesamong glycosyltransferases, and the provision of the novelsialyltransferase of the present invention meets such a high demand.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1-1 shows the results of HPLC analysis obtained for the reactionsolution in which a crude enzyme solution from the strain JT-ISH-224 wasreacted with pyridylaminated lactose (PA-lactose) and CMP-sialic acid.The peaks at retention times of 3.995, 4.389 and 5.396 minutes representPA-lactose, PA-6′-sialyllactose and PA-3′-sialyllactose, respectively.

FIG. 1-2 shows the results of HPLC analysis obtained for the reactionsolution in which a crude enzyme solution from the strain JT-ISH-224 wasreacted with pyridylaminated (PA) lactose. This figure shows the resultsof a control experiment relative to the experiment in FIG. 1-1, in whichCMP-sialic acid was not mixed as a sialic acid donor into the reactionsolution. The peak at a retention time of 3.993 minutes representsPA-lactose.

FIG. 1-3 shows the results of HPLC analysis obtained for a PA-lactosestandard. PA-lactose appears as a peak at a retention time of 4.026minutes.

FIG. 1-3 shows the results of HPLC analysis obtained for aPA-3′-sialyllactose standard. PA-3′-sialyllactose appears as a peak at aretention time of 5.447 minutes.

FIG. 1-5 shows the results of HPLC analysis obtained for the reactionsolution in which a known β-galactoside-α2,6-sialyltransferase derivedfrom Photobacterium damselae strain JT0160 was reacted with PA-lactoseand CMP-sialic acid (i.e., pyridylaminated α2,6-sialyllactose wasproduced). The peaks at retention times of 4.000 and 4.406 minutesrepresent PA-lactose and PA-6′-sialyllactose, respectively.

FIG. 1-6 shows the results of HPLC analysis obtained for the reactionsolution in which a known α2,6-sialyltransferase derived fromPhotobacterium damselae strain JT0160 was reacted with PA-lactose. Thisis a control experiment relative to the experiment in FIG. 1-5, in whichCMP-sialic acid was not mixed into the reaction solution. The peak at aretention time of 3.995 minutes represents PA-lactose.

FIG. 2-1 is a graph showing the effect of reaction pH on the enzymeactivity of JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase N1C0. The abbreviations in thegraph are as follows: Ac: acetate buffer, Cac: cacodylate buffer, Phos:phosphate buffer, and TAPS: TAPS buffer.

FIG. 2-2 is a graph showing the effect of reaction temperature on theenzyme activity of JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase N1C0.

FIG. 2-3 is a graph showing the effect of NaCl concentration in thereaction solution on the enzyme activity of JT-ISH-224-derivedrecombinant β-galactoside-α2,6-sialyltransferase N1C0.

EXAMPLES

The present invention will now be described in more detail by way of thefollowing examples, which are not intended to limit the technical scopeof the invention. Based on the detailed description, modifications andchanges will be apparent to those skilled in the art, and suchmodifications and changes fall within the technical scope of theinvention.

Example 1 Screening and Strain Identification of MicroorganismsProducing β-Galactoside-α2,6-Sialyltransferase

Sea water, sea sand, sea mud or a marine product was used as aninoculum. This inoculum was applied onto agar plates containing marinebroth agar 2216 medium (Becton Dickinson) to obtain microorganismsgrowing at 15° C., 25° C. or 30° C. After the resulting microorganismswere pure-cultured in a routine manner, each microorganism was culturedusing a liquid medium composed of marine broth 2216 medium (BectonDickinson). After the microorganisms were fully grown, the cells werecollected from each culture solution by centrifugation. To the collectedcells, 20 mM cacodylate buffer (pH 6.0) containing 0.2% Triton X-100(Kanto Kagaku, Japan) was added, and the cells were suspended therein.This cell suspension was ultrasonicated under ice cooling to homogenizethe cells. This cell homogenate was used as a crude enzyme solution andmeasured for its sialyltransferase activity, thus obtaining a strainhaving sialyltransferase activity, i.e., JT-ISH-224. Incidentally, thestrain JT-ISH-224 was obtained from the internal organs of barracuda.

Sialyltransferase activity was measured as described in J. Biochem.,120, 104-110 (1996) (which is hereby incorporated by reference in itsentirety). More specifically, the enzymatic reaction was accomplished byusing CMP-NeuAc (70 nmol, containing about 20,000 cpm CMP-NeuAc in whichNeuAc was labeled with ¹⁴C; NeuAc represents N-acetylneuraminic acid) asa glycosyl donor substrate, lactose (1.25 μmol) as a glycosyl acceptorsubstrate, NaCl added to give a concentration of 0.5 M, and theenzyme-containing reaction solution (30 μl) prepared as described above.The enzymatic reaction was carried out at 25° C. for about 10 to 180minutes. After completion of the reaction, 1.97 ml of 5 mM phosphatebuffer (pH 6.8) was added to the reaction solution, which was thenapplied to a Dowex 1×8 (PO₄ ³⁻ form, 0.2×2 cm, BIO-RAD) column. Theradioactivity was measured for the reaction product, i.e., sialyllactosecontained in the eluate (0 to 2 ml) from this column to calculate theenzyme activity. One enzyme unit (1 U) is defined as the amount ofenzyme required to transfer 1 micromole of sialic acid per minute.

To determine the binding mode of sialic acid, a reaction usingPA-lactose as a substrate was then performed. The enzymatic reaction wasaccomplished by using the resulting crude enzyme solution and apyridylaminated sugar chain as a glycosyl acceptor substrate. Thepyridylaminated sugar chain used for analysis was pyridylaminatedlactose (Galβ1-4Glc-PA, Takara Bio Inc., Japan). To 5 μl of the crudeenzyme solution, 1.5 μl of 5 mM CMP-NeuAc and 1.5 μl of 10 μmol/μlglycosyl acceptor substrate were added and reacted at 25° C. for 18hours. After the reaction, the reaction solution was treated at 100° C.for 2 minutes to inactivate the enzyme, followed by HPLC to analyze thereaction product. The HPLC system used was Shimadzu LC10A (ShimadzuCorporation, Japan) and the analytical column used was Takara PALPAKType R (Takara Bio Inc., Japan). The column which had been equilibratedwith 100 mM acetate-triethylamine (pH 5.0) containing 0.15% N-butanolwas injected with the reaction solution supplemented with 72 μl ofEluent A (100 mM acetate-triethylamine, pH 5.0). For elution ofpyridylaminated sugar chains, Eluent A (100 mM acetate-triethylamine, pH5.0) and Eluent B (100 mM acetate-triethylamine containing 0.5%n-butanol, pH 5.0) were used to successively elute the pyridylaminatedsugar chains with a linear gradient of 30% to 50% Eluent B (0 to 20minutes) and then 100% Eluent B (21 to 35 minutes). The analysis wasperformed under the following conditions: flow rate: 1 ml/min, columntemperature: 40° C., detection: fluorescence (Ex: 320 nm, Em: 400 nm).The results indicated that the strain JT-ISH-224 had bothβ-galactoside-α2,6-sialyltransferase activity andβ-galactoside-α2,3-sialyltransferase activity (FIGS. 1-1 to 1-6).

Bacteriological Identification of Strain JT-ISH-224

The resulting strain JT-ISH-224 was found to have the followingproperties:

(Microbiological Properties)

(1) The cells are in bacillary form and have a size of 0.7 to 0.8 μm×1.0to 1.5 μm.

(2) Motility: +

(3) Gram staining: −

(4) Spore: −

(Physiological and Biochemical Properties)

(1) Growth temperature: − at 4° C., + at 25° C., + at 30° C., − at 37°C.

(2) Colony color: not producing characteristic colony pigment

(3) O/F test: +/−

(4) Catalase test: +

(5) − Oxidase test: +

(6) Acid production from glucose: +

(7) Gas generation from glucose: +

(8) Photogenesis: −

(9) Reduction of nitrate: +

(10) Indole formation: +

(11) Glucose acidification: −

(12) Arginine dihydrolase: +

(13) Urease: −

(14) Esculin hydrolysis: −

(15) Gelatin hydrolysis: −

(16) β-Galactosidase: +

(17) Glucose assimilation: −

(18) L-Arabinose assimilation: −

(19) D-Mannose assimilation: −

(20) D-Mannitol assimilation: −

(21) N-Acetyl-D-glucosamine assimilation: −

(22) Maltose assimilation: −

(23) Potassium gluconate assimilation: −(24) n-Capric acid assimilation:−

(25) Adipic acid assimilation: −

(26) dl-Malic acid assimilation: −

(27) Sodium citrate assimilation: −

(28) Phenyl acetate assimilation: −

(29) Cytochrome oxidase: +

(30) O/129 sensitivity: 10 μg −, 15 μg +

(31) GC content of DNA isolated from bacterial cells (mol %): 39.4%

Nucleotide Sequence Analysis of 16S rRNA Gene

The genomic DNA extracted from the strain JT-ISH-224 in a routine mannerwas used as a template for PCR to amplify the entire nucleotide sequenceof the 16S rRNA gene, thereby determining its nucleotide sequence. Thenucleotide sequence is shown in SEQ ID NO: 5.

The strain JT-ISH-224 was shown to belong to the Vibrionaceae, based onits morphological observations including growth on marine agar,bacillary form, Gram staining, fermentative glucose degradation andO/129 sensitivity, along with the results from the physiological andbiochemical property tests. Moreover, the DNA nucleotide sequence of the16S rRNA gene in the strain JT-ISH-224 was found to share the highesthomology (99.2%) with the sequence of the 16S rRNA gene inPhotobacterium phosphoreum the type strain ATCC11040, and the secondhighest homology (99.1%) with the sequence of the 16S rRNA gene inPhotobacterium iliopiscarium the type strain ATCC51760. These resultsindicated that the strain JT-ISH-224 is a microorganism belonging to thegenus Photobacterium of the family Vibrionaceae (Photobacterium sp.).

Example 2 Cloning and Nucleotide Sequencing ofβ-Galactoside-α2,6-Sialyltransferase Gene from Strain JT-ISH-224, and E.coli Expression of the Gene (1) Confirmation of the Presence ofβ-Galactoside-α2,6-Sialyltransferase Gene Homologue in Strain JT-ISH-224

To determine whether there was a homologue of theβ-galactoside-α2,6-sialyltransferase gene derived from Photobacteriumdamselae strain JT0160, genomic Southern hybridization was performed onthe strain JT-ISH-224 that was found to haveβ-galactoside-α2,6-sialyltransferase activity in Example 1. From a cellpellet of the strain JT-ISH-224 (about 0.5 g), genomic DNA (about 100μl) was prepared using a Qiagen Genomic-tip 100/G (Qiagen) in accordancewith the instructions attached to the kit. The genomic DNA (severalmicrograms) from the strain JT-ISH-224 was then digested with arestriction enzyme EcoRI or HindIII and fractionated by 0.7% agarose gelelectrophoresis, followed by alkaline blotting with 0.4 M NaOH totransfer the gel onto a Hybond-N+ nylon membrane filter (AmershamBiosciences). Southern hybridization was performed on this filter usingas a probe a partial fragment (i.e., an EcoRI-HindIII fragment ofapproximately 1.2 kb covering ATG to HindIII) of theβ-galactoside-α2,6-sialyltransferase gene from Photobacterium damselaestrain JT0160 (GeneBank Accession No. E17028). The hybridizationexperiment was performed using an ECL direct labelling & detectionsystem (Amersham). The probe was labeled according to the instructionsattached to the kit. Hybridization was accomplished at 37° C. (generallyat 42° C.) for 4 hours using the hybridization buffer included in thekit, which was supplemented with 5% (w/v) Blocking reagent and 0.5 MNaCl. Washing was performed twice in 0.4% SDS, 0.5×SSC at 50° C.(generally 55° C.) for 20 minutes and once in 2×SSC at room temperaturefor 5 minutes. Signal detection was performed according to theinstructions attached to the kit. As a result, EcoRI digestion detecteda band of approximately 12.5 kb, while HindIII digestion detected a bandof approximately 9 kb. These results indicated that the strainJT-ISH-224 had a homologue of the β-galactoside-α2,6-sialyltransferasegene from Photobacterium damselae strain JT0160.

(2) Cloning of β-galactoside-α2,6-sialyltransferase Gene from StrainJT-ISH-224

(i) Construction of Genomic Library

Relative to 1-2 μg of the genomic DNA from the strain JT-ISH-224, 0.1 to0.2 units of Sau3AI, a four base-cutter enzyme, was used for partialdigestion of the DNA. The genomic DNA was treated in a total amount of80 μg. The reaction buffer used was one attached to the enzyme and thereaction conditions were set at 37° C. for 30 minutes. After thereaction, EDTA (pH 8.0) was added at a final concentration of 25 mM tothe reaction solution, followed by phenol/chloroform treatment. Thegenomic DNA was collected by ethanol precipitation and dissolved in 400μl TE. In a centrifugal tube (Hitachi 40PA), a 40-10% gradient wasprepared from 40% sucrose buffer (20 mM Tris pH 8.0, 5 mM EDTA pH 8.0, 1M NaCl) and 10% sucrose buffer using a gradient preparation unit, andthe above partially-digested DNA solution was overlayed thereon. Usingan ultracentrifuge (Hitachi SCP70H, rotor: SRP28SA), the tube wascentrifuged at 26,000 rpm at 20° C. for 15 hours. After centrifugation,a hole was made with a 25G needle at the bottom of the tube to collectevery 1 ml aliquots from the solution at the bottom. Using a submarineelectrophoretic chamber, a part of each collected sample containing thegenomic DNA was electrophoresed on a 0.5-0.6% agarose gel/TAE buffer at26 V for 20 hours to observe a fraction containing DNA of 9-16 kb size.As a marker, λ/HindIII was used. After addition of 2.5 ml TE to reducethe sucrose concentration, the fraction containing the DNA fragment of9-16 kb size was ethanol precipitated, rinsed and dissolved in a smallvolume of TE.

λDASH II (Stratagene) was used as a vector to create a genomic libraryof the strain JT-ISH-224. The λDASH II/BamHI vector and the genomic DNAfragment were ligated overnight at 12° C. using a Stratagene ligationkit. After the reaction, the reaction solution was reacted with GigaPackIII Gold Packaging extract, whereby the λ vector carrying the genomicDNA was incorporated into phage particles. The phage solution was storedat 4° C. in 500 μl SM buffer and 20 μl chloroform. E. coli XL1-BlueMRA(P2) (Stratagene) was grown in LBMM (LB+0.2% maltose+10 mM MgSO₄) toA₆₀₀=0.5, and 2001 of this culture solution was incubated with anappropriate amount of the phage solution at 37° C. for 15 minutes. Thissolution was mixed with 4 ml NZY top agarose kept at 48° C., and platedin a NZY agar plate (a plastic dish of 9 cm diameter). The plate wascultured overnight at 37° C. and the number of plaques was counted tocalculate the titer. As a result, the library size was calculated to beabout 300,000 pfu (plaque forming unit).

(ii) Plaque Hybridization and Subcloning of Genomic Fragment Containingβ-galactoside-α2,6-Sialyltransferase Gene from Strain JT-ISH-224

Next, the above-mentioned partial fragment of theβ-galactoside-α2,6-sialyltransferase gene from Photobacterium damselaestrain JT0160 was used as a probe to screen the genomic library of thestrain JT-ISH-224. In a round dish of 9 cm diameter, several hundred pfuof phages were plated together with XL1-blue MRA(P2) host cellsaccording to the instructions attached to a λDASH II/BamHI vector kit(Stratagene). Plaques were contacted with a Hybond-N+ nylon membranefilter (Amersham), treated with alkali according to the instructionsattached to the membrane to cause DNA denaturation, and then fixed onthe membrane. Probe labeling and hybridization conditions are asdescribed in (1) above. As a result, 8 clones were obtained up to theend of secondary screening (also serving as plaque purification), 4 ofwhich were collected and each was plated in a NZY plate together with E.coli XL1-blue MRA(P2) at several ten thousand pfu per plate andincubated overnight at 37° C. SM buffer was added in 4 ml volumes to 6plates with confluent plaques, and the plates were allowed to standovernight at 4° C. Phage plate lysates were collected with Pasteurpipettes, and λDNA was extracted and purified from each lysate with aQIAGEN Lambda Mini Kit (QIAGEN). These 4 λDNA samples were digested withrestriction enzymes EcoRI & HindIII, EcoRI & BamHI, or EcoRI & XhoI.Each digest was fractionated by agarose gel electrophoresis andtransferred onto a nylon membrane filter, as described in (1) above.This filter was provided for Southern analysis using as a probe thepartial fragment of the β-galactoside-α2,6-sialyltransferase gene fromPhotobacterium damselae strain JT0160. As a result, EcoRI-BamHIdigestion detected a band of 10 kb. Since a genome of 10 kb in lengthappeared difficult to be subcloned into a high-copy plasmid vector in aroutine manner, Southern hybridization was further performed withvarious restriction enzymes. The enzymes used were BglII, EcoRV, KpnI,NheI, PstI, PvuII, SacI, SalI and XbaI. As a result, EcoRV digestiondetected a band of 6.6 kb, KpnI digestion detected a band of 7 kb, andNheI digestion detected a band of 3.5 kb. Then, each λDNA sample wasdigested again with NheI, followed by agarose gel electrophoresis in TAEbuffer using a low melting point agarose (SeaPlaqueGTG). A DNA fragmentof 3.5 kb was excised as a gel piece, supplemented with an equal volumeof 200 mM NaCl and treated at 65° C. for 10 minutes to dissolve the gel.This sample was extracted once with phenol, once with phenol/chloroform,and then once with chloroform, followed by ethanol precipitation tocollect the 3.5 kb DNA fragment. This fragment was ligated with aLigation kit (Takara Bio Inc., Japan) to a XbaI site of plasmid vectorpBluescript SK(−) which had been dephosphorylated. After ligation, theDNA was transformed into E. coli TB1 by electroporation and plated ontoLA agar medium containing ampicillin (100 μg/mL). After culturingovernight at 37° C., the resulting multiple colonies were inoculatedinto LB medium (containing ampicillin) and cultured overnight withshaking at 37° C., followed by plasmid extraction in a routine manner(Sambrook et al. 1989, Molecular Cloning, A laboratory manual, 2^(nd)edition (hereby incorporated by reference in its entirety)).

(iii) Determination of the Entire Nucleotide Sequence ofβ-galactoside-α2,6-sialyltransferase Gene from Strain JT-ISH-224

Next, nucleotide sequences at both ends of the 3.5 kb NheI fragment weredetermined for the plasmid confirmed above to carry the insert DNA byusing M13 primers (Takara Bio Inc., Japan) in an ABI PRISM fluorescentsequencer (Model 310 Genetic Analyzer, Perkin Elmer). The resulting DNAsequences were translated into amino acid sequences using geneticinformation processing software GENETYX Ver. 7 (Genetyx Corporation,Japan), and an identity search with the BLAST program was made for theseamino acid sequences against the GeneBank database of the NationalCenter for Biotechnology Information (NCBI). As a result, the amino acidsequence translated from one of the DNA sequences showed significantidentity with the amino acid sequence ofβ-galactoside-α2,6-sialyltransferase derived from Photobacteriumdamselae strain JT0160. The orientation of the region showing identitysuggested that the 3.5 kb NheI fragment contained the entireβ-galactoside-α2,6-sialyltransferase gene from the strain JT-ISH-224.

Next, to determined the entire DNA sequence of this enzyme gene from thestrain JT-ISH-224, the following two primers were synthesized based onthe DNA sequence obtained from the 3.5 kb NheI fragment, and used fornucleotide sequencing:

ISH224-26ST-C3-R (5′-TTCATCGTCATCTAATCGTGGC-3′ (22 mer): SEQ ID NO: 6);and ISH224-26ST-C4-R (5′-AGTTGTTGCGTACCACAAGT-3′ (20 mer): SEQ ID NO:7).

Using these primers, nucleotide sequencing was performed as describedabove. As a result, the sequence of SEQ ID NO: 1 in the Sequence Listingwas obtained. This sequence corresponds to the entire nucleotidesequence of the open reading frame (ORF) of theβ-galactoside-α2,6-sialyltransferase gene from the strain JT-ISH-224.The ORF of the β-galactoside-α2,6-sialyltransferase gene fromPhotobacterium sp. strain JT-ISH-224 was composed of 1545 base pairs andencoded 514 amino acids. This amino acid sequence is shown in SEQ ID NO:2 in the Sequence Listing. Upon analysis of DNA and amino acid sequencesusing GENETYX Ver. 7, the DNA sequence of theβ-galactoside-α2,6-sialyltransferase gene from the strain JT-ISH-224 wasfound to share an identity of 63% with theβ-galactoside-α2,6-sialyltransferase gene from Photobacterium damselaestrain JT0160. Likewise, its amino acid sequence was found to share anidentity of 54.5% with β-galactoside-α2,6-sialyltransferase (JC5898)from Photobacterium damselae strain JT0160.

(3) Construction of Expression Vector forβ-Galactoside-α2,6-Sialyltransferase Gene from Strain JT-ISH-224

To test whether the cloned gene had sialyltransferase activity or toobtain β-galactoside-α2,6-sialyltransferase derived from the strainJT-ISH-224 in large amounts, the full length of the gene and itsderivative modified to remove the N-terminal signal peptide region wereeach integrated into an expression vector to produce a protein in E.coli cells, followed by measuring the activity of this expressedprotein.

Genetic information processing software GENETYX Ver. 7 was used toanalyze the amino acid sequence of β-galactoside-α2,6-sialyltransferasederived from the strain JT-ISH-224, estimating that the N-terminal 17amino acids would constitute the signal peptide. Then, primers forcloning the full-length gene (herein referred to as “ISH224-N0C0”):ISH224-26ST-N0BspHI (5′-AGAATATCATGAAAAACTTTTTATTATTAAC-3′ (31 mer): SEQID NO: 8) and ISH224-26ST-COBamHI(5′-TTTTTTGGATCCCTAGACTGCAATACAAACACC-3′ (33 mer): SEQ ID NO: 10), aswell as primers for cloning a gene encoding a protein lacking the aminoacids of the signal peptide region (herein referred to as“ISH224-N1C0”): ISH224-26ST-NlPciI(5′-CTTGTAACATGTCAGAAGAAAATACACAATC-3′ (31 mer): SEQ ID NO: 9) andISH224-26ST-COBamHI (5′-TTTTTTGGATCCCTAGACTGCAATACAAACACC-3′ (33 mer):SEQ ID NO: 10) were designed and synthesized.

PCR was carried out with these primers using the plasmid carrying the3.5 kb NheI fragment as a template to amplify theβ-galactoside-α2,6-sialyltransferase gene from the strain JT-ISH-224 foruse in integration into an expression vector. The reaction conditionsfor PCR were set as follows. In 50 μl reaction solution containing 500ng template DNA, 5 μl 10× Ex taq buffer, 4 μl 2.5 mM dNTPs, 50 pmolprimer and 0.5 μl Ex taq (Takara Bio Inc., Japan), PCR was carried outusing a Program Temp Control System PC-700 (ASTEK) under the followingconditions: 96° C. for 3 minutes, (96° C. for 1 minute, 55° C. for 1minute, 72° C. for 2 minutes)×5 cycles, and 72° C. for 6 minutes. As aresult, PCR products of approximately 1.55 kb and 1.5 kb were amplifiedfor ISH224-N0C0 and ISH224-N1C0, respectively. These PCR products wereeach cloned into vector pCR4TOPO (Invitrogen). Ligation was carried outaccording to the instructions attached to the vector kit. Each DNA wasintroduced into E. coli TB1 by electroporation and the plasmid DNA wasextracted in a routine manner (Sambrook et al. 1989, Molecular Cloning,A laboratory manual, 2^(nd) edition). Clones confirmed to have theinsert were each analyzed by PCR with M13 primers (Takara Bio Inc.,Japan) to determine the nucleotide sequence of the PCR product from bothends using an ABI PRISM fluorescent sequencer (Model 310 GeneticAnalyzer, Perkin Elmer). As a result, ISH224-N0C0 had a nucleotidesubstitution from thymine (T) to cytosine (C) at position 718 of SEQ IDNO: 1 in the Sequence Listing. This mutation results in a codon changefrom TTA to CTA, but causes no amino acid mutation because these codonsboth encode leucine (Leu). On the other hand, ISH224-N1C0 had nomutation in its nucleotide sequence. The nucleotide sequence ofISH224-N1C0 is shown in SEQ ID NO: 3.

One selected clone of ISH224-N0C0 or ISH224-N1C0 whose nucleotidesequence was confirmed was double-digested with restriction enzymesBspHI & BamHI (for ISH224-N0C0) or PciI & BamHI (for ISH224-N1C0),followed by gel purification as described in (2)(ii) above. pTrc99A(Pharmacia LKB) was used as a vector for E. coli expression. After beingdouble-digested with restriction enzymes NcoI & BamHI and purified on agel, this vector was ligated with the restriction enzyme-treated PCRproduct of ISH224-N0C0 or ISH224-N1C0 using a Takara Ligation Kit(Takara Bio Inc., Japan) and transfected into E. coli TB1. In a routinemanner, the plasmid DNA was extracted and analyzed by restriction enzymeanalysis to confirm the integration of the insert, thereby completingISH224-N0C0/pTrc or ISH224-N1C0/pTrc.

Further, to create various truncated proteins ofβ-galactoside-α2,6-sialyltransferase derived from the strain JT-ISH-224,the following primers were designed.

Primer Name, Sequence (SEQ ID NO), Length

224-26-N2Bsp AAACTTTCATGACGCAACAACTATTAACAGAA (SEQ ID NO: 15), 32 mer224-26-N3Bsp AAGTAATCATGAACGTAGTGGCTCCATCTTTA (SEQ ID NO: 16), 32 mer224-26-N3.1Bsp CACGTGTCATGACTCTTCAGCAGCTAATGGAT (SEQ ID NO: 17), 32 mer

224-26-N2Bsp allows deletion of N-terminal 62 amino acids andintroduction of methionine at the N-terminus.

224-26-N3Bsp allows deletion of N-terminal 110 amino acids andintroduction of methionine at the N-terminus. 224-26-N3.1Bsp allowsdeletion of N-terminal 127 amino acids and introduction of methionine atthe N-terminus. Using these primers in combination with primerISH224-26ST-COBamHI shown above, PCR was carried out as described aboveusing ISH224-N1C0 as a template. The resulting PCR products were eachcloned into vector pCR4TOPO (Invitrogen) and confirmed for theirnucleotide sequence. As a result, in all clones obtained with theseprimer combinations, their nucleotide sequences were found to share 100%homology with the nucleotide sequence of ISH224-N1C0 used as a template.The clone obtained from a combination of 224-26-N2Bsp andISH224-26ST-COBamHI was designated as N2C0. Likewise, the clone obtainedfrom a combination of 224-26-N3Bsp and ISH224-26ST-COBamHI wasdesignated as N3C0, while the clone obtained from a combination of224-26-N3.1Bsp and ISH224-26ST-COBamHI was designated as N3.1C0. Afterbeing double-digested with restriction enzymes BspHI & BamHI, theseclones were each cloned into an E. coli expression vector (pTrc99A) asdescribed above to thereby complete ISH224-N2C0/pTrc, ISH224-N3C0/pTrcand ISH224-N3.1C0/pTrc.

(4) Expression Induction and Activity Measurement

An induction experiment of protein expression was performed on the fiveclones obtained in (3) above (i.e., ISH224-N0C0/pTrc, ISH224-N1C0/pTrc,ISH224-N2C0/pTrc, ISH224-N3C0/pTrc and ISH224-N3.1C0/pTrc). A singlecolony of E. coli TB1 having the expression vector pTrc99A carrying eachclone was inoculated into LB medium (6 ml) containing an antibiotic,ampicillin (final concentration 100 μg/mL), and pre-cultured at 30° C.to about A₆₀₀=0.5, followed by addition of IPTG(isopropyl-β-D(−)-thiogalactopyranoside, Wako Pure Chemical Industries,Ltd., Japan) at a final concentration of 1 mM. After culturing withshaking at 30° C. for an additional 4 hours, the cells in 4 ml culturesolution were collected by centrifugation. These cells were suspended in200 μl of 20 mM Bis-Tris buffer (pH 7.0) containing 0.336% Triton X-100and 0.5 M sodium chloride, and ultrasonically homogenized under icecooling. The resulting homogenate was defined as a crude enzymesolution, diluted 200-fold with 20 mM cacodylate buffer (pH 5.0)containing 0.336% Triton X-100, and then provided for activitymeasurement. The reaction was carried out in duplicate. The reactionconditions are as indicated in the footnotes of Tables 1-1 and 1-2. As aresult, as shown in Tables 1-1 and 1-2 below, it was demonstrated thatthere was a factor transferring ¹⁴C-labeled NeuAc in the glycosyl donorCMP-NeuAc to the glycosyl acceptor substrate lactose, i.e.,sialyltransferase activity in the crude enzyme solutions from all clonesbut ISH224-N3.1C0/pTrc. These results indicated that E. coli cells intowhich ISH224-N0C0/pTrc, ISH224-N1C0/pTrc, ISH224-N2C0/pTrc orISH224-N3C0/pTrc had been introduced expressed sialyltransferase.

In view of the foregoing, ISH224-N3C0 was found to be the smallest clonethat retained the activity of β-galactoside-α2,6-sialyltransferasederived from the strain JT-ISH-224. Thus, it was indicated that thepresence of at least amino acids 111-514 of SEQ ID NO: 2 allowedretention of β-galactoside-α2,6-sialyltransferase activity.

TABLE 1-1 Sialyltransferase activity in homogenate of E. coli into whichβ-galactoside-α2,6-sialyltransferase gene from strain JT-ISH-224 isintroduced Radioactivity (cpm) Crude enzyme solution Round 1 Round 2Average ISH224-N0C0 clone 2859 2617 2738 ISH224-N1C0 clone 1552 17111631.5 Absence 94 113 103.5

Reaction Conditions

Reaction composition:

3 M NaCl 5 μl 45 mg/ml Lactose (in 20 mM cacodylate buffer (pH 5)) 10μl  Crude enzyme solution diluted 200-fold 5 μl 4.55 mM CMP-sialic acid(in 20 mM cacodylate buffer 5 μl (pH 5)) + ¹⁴C-CMP-sialic acid Reactiontime: 2 minutes Reaction temperature: 30° C.

TABLE 1-2 Sialyltransferase activity in homogenate of E. coli into whichβ-galactoside-α2,6-sialyltransferase gene from strain JT-ISH-224 isintroduced Crude enzyme solution Radioactivity ISH224-N0C0 clone +ISH224-N1C0 clone + ISH224-N2C0 clone + ISH224-N3C0 clone +ISH224-N3.1C0 clone − Absence −

Reaction Conditions Reaction Composition:

3M NaCl 5 μl 45 mg/ml Lactose (in 20 mM cacodylate buffer (pH 5)) 10 μl Crude enzyme solution diluted 200-fold 5 μl 4.55 mM CMP-sialic acid (in20 mM cacodylate buffer 5 μl (pH 5)) + ¹⁴C-CMP-sialic acid Reactiontime: 2 minutes Reaction temperature: 30° C.

(5) Confirmation of β-Galactoside-α2,6-Sialyltransferase Activity

The crude enzyme solution prepared in (4) above from the ISH224-N1C0 orISH224-N0C0 clone was used to examine whether sialyltransferaseexpressed by E. coli cells into which ISH224-N0C0/pTrc orISH224-N1C0/pTrc had been introduced hadβ-galactoside-α2,6-sialyltransferase activity. As in the case of Example1, pyridylaminated lactose (Galβ1-4Glc-PA, PA-Sugar Chain 026, TakaraBio Inc., Japan) was used as a glycosyl acceptor to carry out theenzymatic reaction. As a result, PA-6′-sialyllactose(Neu5Acα2-6Galβ1-4Glc-PA) was detected, as in the case of Example 1.Namely, sialyltransferases derived from both clone strains were found tohave β-galactoside-α2,6-sialyltransferase activity. These resultsdemonstrated that the β-galactoside-α2,6-sialyltransferase gene fromPhotobacterium sp. strain JT-ISH-224 was cloned and expressed in E. colicells.

Example 3 Productivity of Recombinantβ-Galactoside-α2,6-Sialyltransferase Derived from JT-ISH-224 Comparisonbetween ISH224-N0C0 and ISH224-N1C0 Clones

A time-dependent induction experiment of protein expression wasperformed on the ISH224-N0C0 and ISH224-N1C0 clones obtained in Example2. A single colony of E. coli TB1 having the expression vector pTrc99Acarrying each clone was inoculated into LB medium (6 ml) containing anantibiotic, ampicillin (final concentration 100 μg/mL), and pre-culturedat 30° C. for about 8 hours. This pre-cultured solution was inoculatedinto LB medium (300 ml) containing and antibiotic, ampicillin (finalconcentration 100 μg/mL) and cultured with shaking at 30° C. When OD600reached around 0.5, IPTG (isopropyl-β-D(−)-thiogalactopyranoside, WakoPure Chemical Industries, Ltd., Japan) was added at a finalconcentration of 1 mM, followed by culturing with shaking at 30° C. At4, 6, 22 and 28 hours after culturing, the cells in each culturesolution were collected by centrifugation. These cells were suspended in20 mM Bis-Tris buffer (pH 6.0) containing 0.336% Triton X-100, andultrasonically homogenized under ice cooling. The resulting homogenatewas defined as a crude enzyme solution, diluted 200-fold with 20 mMcacodylate buffer (pH 5.0) containing 0.336% Triton X-100, and thenprovided for activity measurement. The reaction was carried out induplicate. The reaction conditions are as indicated in the footnote ofTable 2. As a result, as shown in Table 2 below, the ISH224-N0C0 cloneshowed maximum β-galactoside-α2,6-sialyltransferase activity at 4 hoursafter IPTG addition, and its productivity was 5,501 U/L of medium. Onthe other hand, the ISH224-N1C0 clone showed maximumβ-galactoside-α2,6-sialyltransferase activity at 22 hours after IPTGaddition, and its productivity was 10,776 U/L of medium.

TABLE 2 Sialyltransferase activity in homogenate of E. coli into whichβ-galactoside-α2,6- sialyltransferase gene from strain JT-ISH-224 isintroduced Amount Buffer of Enzyme Hours volume transferredconcentration in Total after Culture used for sialic crude enzyme enzymeProductivity Clone IPTG volume homogenization Radioactivity acidsolution activity (U/L of name addition (L) (ml) (CPM) (nmol) (unit/ml)(unit) medium) ISH224- 4 0.3 28.3 1235.5 1.46 58.3 1650 5501 N0C0 6 0.326.0 791.0 0.93 37.3 971 3236 22 0.3 30.9 638.0 0.75 30.1 931 3102 280.3 32.0 512.0 0.60 24.2 773 2578 ISH224- 4 0.3 26.0 142.5 0.17 6.8 177589 N1C0 6 0.3 26.0 311.0 0.37 14.8 385 1285 22 0.3 37.5 1809.0 2.1686.2 3233 10776 28 0.3 38.6 1459.0 1.74 69.5 2684 8946

Reaction Conditions Reaction Composition:

3 M NaCl 5 μl 45 mg/ml Lactose (in 20 mM cacodylate buffer (pH 5)) 10μl  Crude enzyme solution diluted 200-fold 5 μl 4.55 mM CMP-sialic acid(in 20 mM cacodylate buffer 5 μl (pH 5)) + ¹⁴C-CMP-sialic acid Reactiontime: 1 minute Reaction temperature: 30° C.

These results indicated that the productivity ofβ-galactoside-α2,6-sialyltransferase was higher in the ISH224-N1C0 clonethan in the ISH224-N0C0 clone.

As has been shown before the filing of the present application,Photobacterium damselae-derived recombinantβ-galactoside-α2,6-sialyltransferase, i.e.,β-galactoside-α2,6-sialyltransferase produced by E. coli cellstransformed with plasmid pEBSTA178 has a productivity of 224.5 U/L(Yamamoto, T., et al., J. Biochem., 120, 104-110 (1996)). When comparedto this enzyme, JT-ISH-224-derived recombinant α2,6-sialyltransferasewas found to have about 48-fold higher productivity. Likewise, as amicrobial sialyltransferase, Pasteurella multocida-derivedα2,3-sialyltransferase is known to have a high productivity of 6,000 U/L(Yu, H. et al., J. Am. Chem. Soc., 127, 17618-17619, 2005), although itis categorized as a different type of enzyme. When compared to thisenzyme, JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase was also found to have about1.8-fold higher productivity.

Example 4-1 Extraction and Purification ofβ-galactoside-α2,6-sialyltransferase from ISH224-N1C0, andAmino-Terminal Amino Acid Sequencing of the Purified Protein (1)Extraction and Purification

From colonies of ISH224-N1C0 subcultured on LBAmp agar plates, the cellswere collected with a loop, inoculated into 6 ml-LB liquid medium (10ml) supplemented with 30 μl of ×200 ampicillin (400 mg/20 ml), andcultured with shaking at 30° C. at 180 rpm for 8 hours.

Main culturing was accomplished in the following manner. 300 ml-LBmedium supplemented with 1.5 ml of ×200 ampicillin (400 mg/20 ml) and300 μl of 1M IPTG (1.192 g/5 ml) was charged into a 1000 ml baffleflask. The same medium was prepared in 9 flasks (2.7 L in total). Eachflask was inoculated with the above culture solution (12 ml) andcultured with shaking at 30° C. at 180 rpm for 24 hours. The culturedmedium was centrifuged to collect the cells (about 15 g on a wet weightbasis).

The cells were suspended in 990 ml of 20 mM Bis-Tris buffer (pH 6.0)containing 0.336% Triton X-100 to give a concentration of 1.6 g/26 ml,and ultrasonically homogenized under ice cooling. The cell homogenatewas centrifuged at 4° C. at 100,000×g for 1 hour to obtain thesupernatant.

This crude enzyme solution was loaded to an anion exchange column calledHiLoad 26/10 Q Sepharose HP (Amersham), which had been equilibrated with20 mM Bis-Tris buffer (pH 6.0) containing 0.336% Triton X-100. Thecolumn was eluted with a linear gradient up to 1 M sodium chloride in 20mM Bis-Tris buffer (pH 6.0) containing 0.336% Triton X-100 to therebycollect an enzymatically active fraction eluted at around 0.25 M sodiumchloride concentration.

The collected fraction was diluted with 20 mM phosphate buffer (pH 6.0)and loaded to hydroxyapatite (Bio-Rad) which had been equilibrated with20 mM phosphate buffer (pH 6.0) containing 0.336% Triton X-100, followedby elution with a linear gradient from 20 mM phosphate buffer (pH 6.0)containing 0.336% Triton X-100 to 500 mM phosphate buffer (pH 6.0)containing 0.336% Triton X-100 to thereby collect an enzymaticallyactive fraction eluted at around 125 mM phosphate buffer concentration.

This fraction was loaded to a MonoQ 5/50 GL (Amersham) anion exchangecolumn. The column was eluted with a linear gradient up to 1 M sodiumchloride in 20 mM Bis-Tris buffer (pH 6.0) containing 0.336% TritonX-100 to thereby collect an enzymatically active fraction eluted ataround 300 mM sodium chloride concentration.

The active fraction was electrophoresed on an SDS-polyacrylamide gel(the concentration of the acrylamide gel: 12.5%), indicating that thetarget enzyme showed a single band with a molecular weight of about56,000. The specific activity of this purified fraction was about9.4-fold higher than that of the cell homogenate (Table 3-1). Theseresults indicated that JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase N1C0 had a specific activity of 113U/mg, which was about 21-fold higher than that (5.5 U/mg) ofβ-galactoside-α2,6-sialyltransferase derived from Photobacteriumdamselae strain JT0160 (J. Biochem., 120, 104-110, 1996, T. Yamamoto etal.). Likewise, Pasteurella multocida-derivedβ-galactoside-α2,3-sialyltransferase has a specific activity of 60 U/mg(Yu, H. et al., J. Am. Chem. Soc., 127, 17618-17619, 2005.), although itis categorized as a different type of enzyme. When compared to thisenzyme, JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase N1C0 is also found to have about1.9-fold higher specific activity.

As to purification of β-galactoside-α2,6-sialyltransferase of theISH224-N1C0 clone from a crude enzyme solution, Table 3-1 shows theenzyme activity of the sample after each of the purification stepsmentioned above. The enzyme activity was measured by the method reportedin J. Biochem. 120, 104-110 (1996), in the same manner as described inExample 1 under conditions as indicated in the footnote of Table 3-1.For protein quantification, a Coomassie Protein Assay Reagent (PIERCE)was used according to the instruction manual attached thereto. Oneenzyme unit (1 U) was defined as the amount of enzyme required totransfer 1 micromole of sialic acid per minute.

TABLE 3-1 Purification of β-galactoside-α2,6-sialyltransferase derivedfrom strain ISH224-N1C0 from crude enzyme solution Total Total SpecificPurification Volume protein activity activity degree Purification step(ml) (mg) (U) (U/mg) Yield (%) (fold) Crude enzyme solution 237.5 1,10013167 12.0 100 1.0 Q sepharose 64.0 371 11794 31.8 90 2.7 Hydroxyapatite180.0 138 9448 68.3 72 5.7 Mono Q 4.5 24.1 2720 113 21 9.4

Reaction Conditions Reaction Composition:

3 M NaCl 5 μl 360 mM Lactose 10 μl  1 M Bis-Tris buffer (pH 6) 3 μlWater 2 μl Enzyme solution 5 μl 14 mM CMP-sialic acid (in 20 mM Bis-Trisbuffer 5 μl (pH 6)) + ¹⁴C-CMP-sialic acid Reaction time: 5 minutesReaction temperature: 25° C.

(2) Amino-Terminal Amino Acid Sequencing

The enzyme solution purified to a single band in (1) above waselectrophoresed on an SDS-polyacrylamide gel (the concentration of theacrylamide gel: 12.5%). After electrophoresis, the protein wastransferred onto a PVDF membrane and stained with CBB. A band region ofinterest was then excised and analyzed for its amino acid sequence witha Procise 494 HT Protein Sequencing System (Applied Biosystems). As aresult, a sequence whose amino-terminus started with serine wasdetermined up to the 15th residue (Ser Glu Glu Asn Thr Gln Ser Ile IleLys Asn Asp Ile Asn Lys). This result suggests that in theβ-galactoside-α2,6-sialyltransferase protein produced by E. coli cellstransformed with the ISH224-N1C0 clone, its amino-terminal methioninewas processed within the E. coli cells.

Example 4-2 Extraction and Purification ofβ-galactoside-α2,6-sialyltransferase from ISH224-N3C0 (1) Extraction andPurification

From colonies of ISH224-N3C0 subcultured on LBAmp agar plates, the cellswere collected with a loop, inoculated into 6 ml-LB liquid medium (10ml) supplemented with 30 μl of ×200 ampicillin (400 mg/20 ml), andcultured with shaking at 30° C. at 180 rpm for 8 hours.

Main culturing was accomplished in the following manner. 300 ml-LBmedium supplemented with 1.5 ml of ×200 ampicillin (400 mg/20 ml) and300 μl of 1M IPTG (1.192 g/5 ml) was charged into a 1000 ml baffleflask. The same medium was prepared in 18 flasks (5.4 L in total). Eachflask was inoculated with the above culture solution (12 ml) andcultured with shaking at 30° C. at 180 rpm for 24 hours. The culturedmedium was centrifuged to collect the cells (about 33.1 g on a wetweight basis).

The cells were suspended in 538.5 ml of 20 mM Bis-Tris buffer (pH 6.0)containing 0.336% Triton X-100 to give a concentration of 1.6 g/26 ml,and ultrasonically homogenized under ice cooling. The cell homogenatewas centrifuged at 4° C. at 100,000×g for 1 hour to obtain thesupernatant.

This crude enzyme solution was loaded to an anion exchange column calledHiLoad 26/10 Q Sepharose HP (Amersham), which had been equilibrated with20 mM Bis-Tris buffer (pH 6.0) containing 0.336% Triton X-100. Thecolumn was eluted with a linear gradient up to 1 M sodium chloride in 20mM Bis-Tris buffer (pH 6.0) containing 0.336% Triton X-100 to therebycollect an enzymatically active fraction eluted at around 0.25 M sodiumchloride concentration.

The collected fraction was diluted with 20 mM phosphate buffer (pH 6.0)and loaded to hydroxyapatite (Bio-Rad) which had been equilibrated with20 mM phosphate buffer (pH 6.0) containing 0.336% Triton X-100, followedby elution with a linear gradient from 20 mM phosphate buffer (pH 6.0)containing 0.336% Triton X-100 to 500 mM phosphate buffer (pH 6.0)containing 0.336% Triton X-100 to thereby collect an enzymaticallyactive fraction eluted at around 125 mM phosphate buffer concentration.This fraction was loaded to a MonoQ 5/50 GL (Amersham) anion exchangecolumn. The column was eluted with a linear gradient up to 1 M sodiumchloride in 20 mM Bis-Tris buffer (pH 6.0) containing 0.336% TritonX-100 to thereby collect an enzymatically active fraction eluted ataround 300 mM sodium chloride concentration.

The active fraction was electrophoresed on an SDS-polyacrylamide gel(the concentration of the acrylamide gel: 12.5%), indicating that thetarget enzyme showed a single band with a molecular weight of about40,000. The specific activity of this purified fraction was about131.5-fold higher than that of the cell homogenate (Table 3-2). Theseresults indicated that JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase N3C0 had a specific activity of 264U/mg, which was about 48-fold higher than that (5.5 U/mg) ofβ-galactoside-α2,6-sialyltransferase derived from Photobacteriumdamselae strain JT0160 (J. Biochem., 120, 104-110, 1996, T. Yamamoto etal.). Likewise, Pasteurella multocida-derivedβ-galactoside-α2,3-sialyltransferase has a specific activity of 60 U/mg(Yu, H. et al., J. Am. Chem. Soc., 127, 17618-17619, 2005.), although itis categorized as a different type of enzyme. When compared to thisenzyme, JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase N3C0 is also found to have about4.4-fold higher specific activity.

As to purification of β-galactoside-α2,6-sialyltransferase N3C0 of theISH224-N3C0 clone from a crude enzyme solution, Table 3-2 shows theenzyme activity of the sample after each of the purification stepsmentioned above. The enzyme activity was measured by the method reportedin J. Biochem. 120, 104-110 (1996), in the same manner as described inExample 1 under conditions as indicated in the footnote of Table 3-2.For protein quantification, a Coomassie Protein Assay Reagent (PIERCE)was used according to the instruction manual attached thereto. Oneenzyme unit (1 U) was defined as the amount of enzyme required totransfer 1 micromole of sialic acid per minute.

TABLE 3-2 Purification of β-galactoside-α2,6-sialyltransferase derivedfrom strain ISH224-N3C0 from crude enzyme solution Total Total SpecificPurification Volume protein activity activity degree Purification step(ml) (mg) (U) (U/mg) Yield (%) (fold) Crude enzyme solution 505.0 24454911 2.0 100 1.0 Q sepharose 39.0 136 3614 26.5 74 13.2 Hydroxyapatite6.0 6.6 997 151 20 75.4 Mono Q 1.5 3.1 808 264 16 131.5

Reaction Conditions Reaction Composition:

3 M NaCl 5 μl 360 mM Lactose 10 μl  1 M Cacodylate buffer (pH 5) 3 μlWater 2 μl Enzyme solution 5 μl 14 mM CMP-sialic acid (in 20 mMcacodylate buffer 5 μl (pH 5)) + ¹⁴C-CMP-sialic acid Reaction time: 5minutes Reaction temperature: 30° C.

Example 5 Optimum pH, Optimum Temperature and Optimum Salt Concentrationfor Enzyme Activity of Recombinant β-galactoside-α2,6-sialyltransferaseN1C0 Derived from JT-ISH-224

The purified enzyme prepared in Example 4-1 was used to examine theoptimum pH, optimum temperature and optimum salt concentration forJT-ISH-224-derived recombinant β-galactoside-α2,6-sialyltransferaseN1C0.

(1) Optimum pH for Enzyme Activity of JT-ISH-224-Derived Recombinantβ-Galactoside-α2,6-Sialyltransferase N1C0

Acetate buffer (pH 4.0, pH 4.5 and pH 5.0), cacodylate buffer (pH 5.0,pH 5.5, pH 6.0, pH 6.5 and pH 7.0), phosphate buffer (pH 7.0, pH 7.5 andpH 8.0) and TAPS buffer (pH 8.0, pH 8.5 and pH 9.0) were prepared andused for enzyme activity measurement at 25° C. at various pH values.

As a result, as shown in FIG. 2-1, the enzyme activity was maximum at pH5.0. It should be noted that enzyme activity at each pH was expressed asrelative activity, assuming that the enzyme activity at pH 5.0 was setto 100.

(2) Optimum Temperature for Enzyme Activity of JT-ISH-224-DerivedRecombinant β-Galactoside-Q2,6-Sialyltransferase N1C0

The enzyme activity was measured at an interval of 5° C. starting from10° C. up to 50° C. using cacodylate buffer (pH 5.0).

As a result, as shown in FIG. 2-2, the enzyme activity was maximum at30° C. It should be noted that enzyme activity at each temperature wasexpressed as relative activity, assuming that the enzyme activity at 30°C. was set to 100.

(3) Optimum Salt Concentration for Enzyme Activity of JT-ISH-224-DerivedRecombinant β-Galactoside-α2,6-Sialyltransferase N1C0

The enzyme activity was measured at 30° C. using cacodylate buffer (pH5.0) by adjusting the NaCl concentration in the reaction solution to 0M, 0.1 M, 0.25 M, 0.5 M, 0.75 M, 1.0 M, 1.5 M or 2.0 M.

As a result, as shown in FIG. 2-3, the enzyme activity was maximumbetween 0.5 M and 0.75 M. Moreover, the enzyme activity was maintainedat substantially the same level between 0 M and 1.0 M. It should benoted that enzyme activity at each NaCl concentration was expressed asrelative activity, assuming that the enzyme activity at 0 M NaClconcentration was set to 100.

Example 6 Comparison of Acceptor Substrate (Monosaccharide, Disaccharideand Trisaccharide) Specificity Between JT-ISH-224-Derived Recombinantβ-Galactoside-α2,6-Sialyltransferase and Strain JT0160-Derivedβ-Galactoside-α2,6-Sialyltransferase (Known Enzyme) (Material andMethod)

Cell homogenates prepared from E. coli cells into whichJT-ISH-224-derived N1C0 had been introduced and from Photobacteriumdamselae strain JT0160 were each purified by ion exchange chromatographyand hydroxyapatite chromatography to give an electrophoretically singleband. The thus purified β-galactoside-α2,6-sialyltransferases were usedin the following experiment in order to examine the presence or absenceof sialyltransferase activity toward various monosaccharides,disaccharides and trisaccharides.

Sialic Acid Transfer Reaction Using Various Glycosyl Acceptor Substrates

A reaction solution (24 μl) was prepared to containCMP-¹⁴C-NeuAc-containing CMP-NeuAc as a glycosyl donor substrate (10.9nmol (8485 cpm), final concentration in the reaction solution: 0.455mM), any of various glycosyl acceptor substrates dissolved in 20 mMcacodylate buffer (pH 5.0) (1 μmol, final concentration in the reactionsolution: 42 mM), sialyltransferase (3.0 mU for JT-ISH-224-derived N1C0,4.3 mU for JT0160) and NaCl (final concentration in the reactionsolution: 500 mM), and reacted at 30° C. for 2 minutes or 60 minutes.The monosaccharides used as glycosyl acceptor substrates were thefollowing 8 types: methyl-α-D-galactopyranoside (Gal-α-OMe),methyl-β-D-galactopyranoside (Gal-β-OMe), methyl-α-D-glucopyranoside(Glc-α-OMe), methyl-β-D-glucopyranoside (Glc-β-OMe),methyl-α-D-mannopyranoside (Man-α-OMe), methyl-β-D-mannopyranoside(Man-β-OMe), N-acetylgalactosamine (GalNAc), and N-acetylglucosamine(GalNAc). The disaccharides used were the following 5 types: lactose(Gal-β1,4-Glc), N-acetyllactosamine (Gal-β1,4-GlcNAc),methyl-β-D-galactopyranosyl-β1,3-N-acetylglucosaminide(Gal-β1,3-GlcNAc-β-OMe),methyl-α-D-galactopyranosyl-α1,3-galactopyranoside (Gal-α1,3-Gal-α-OMe),and methyl-β-D-galactopyranosyl-β-1,3-galactopyranoside(Gal-β1,3-Gal-β-OMe). The trisaccharide used was 2′-fucosyllactose(Fuc-α1,2-Galβ1,4-Glc). It should be noted that the final concentrationwas set to 8.4 mM for the sugar chains shown in Table 4-2, i.e.,methyl-α-D-galactopyranosyl-α3-galactosaminide (Gal-α1,3-Gal-α-OMe),methyl-β-D-galactopyranosyl-β1,3-galactosaminide (Gal-β1,3-Gal-β-OMe)and 2′-fucosyllactose (Fuc-α1,2-Galβ1,4-Glc).

After completion of the enzymatic reaction, 1.98 ml of 5 mM phosphatebuffer (pH 6.8) was added to the reaction solution to stop the enzymaticreaction. Then, the enzymatic reaction solution was diluted with 5 mMphosphate buffer (Ph 6.8) and applied in a volume of 2 ml to aAG1-×2Resin (PO₄ ³⁻ form, 0.2×2 cm) column. This column was prepared asfollows: AG1-×2Resin (OH⁻ form, BIO-RAD) was suspended in 1 M phosphatebuffer (pH 6.8), and after 30 minutes, the resin was washed withdistilled water and then suspended in distilled water. The eluate (0 to2 ml) from this column was measured for its radioactivity. The eluatefrom this column contains the unreacted glycosyl acceptor substrate andthe ¹⁴C-NeuAc (N-acetylneuraminic acid)-bound reaction product which wasgenerated by the reaction, whereas unreacted CMP-¹⁴C-NeuAc is stillretained on the column. Thus, the radioactivity of ¹⁴C from each sialicacid-containing sugar chain generated as a result of the enzymaticreaction arises exclusively from the reaction product, so that theradioactivity of this fraction can be used to calculate the enzymeactivity.

In the manner described above, the radioactivity of NeuAc transferred toeach glycosyl acceptor substrate was measured to calculate the amount oftransferred sialic acid.

(Results)

Sialic acid was found to be transferred to all the 14 monosaccharides,disaccharides and trisaccharide used as glycosyl acceptor substrates inthis experiment (Tables 4-1 and 4-2). JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase showed high glycosyltransferaseactivity over a wide range of acceptor substrates, when compared to theknown β-galactoside-α2,6-sialyltransferase derived from the strainJT0160. More specifically, JT-ISH-224-derived recombinantβ-galactoside-α2,6-sialyltransferase N1C0 showed higher activity thanthe known β-galactoside-α2,6-sialyltransferase derived from the strainJT0160 in the following 6 glycosyl acceptors:methyl-β-D-galactopyranoside, N-acetylgalactosamine,N-acetyllactosamine,methyl-β-D-galactopyranosyl-1,3-N-acetylglucosaminide,methyl-β-D-galactopyranosyl-1,3-galactopyranoside and 2′-fucosyllactose.Thus, this recombinant enzyme was found to have a wider range ofacceptor substrate specificity. It should be noted that the relativeactivity toward each acceptor substrate was calculated assuming that thesialyltransferase activity toward lactose was set to 100.

TABLE 4-1 Acceptor substrate specificity ofβ-galactoside-α2,6-sialyltransferase derived from strain ISH224-N1C0(No. 1) Acceptor substrate JT-ISH-224-N1C0 JT0160 SialyltransferaseSialyltransferase activity activity Relative Relative activity activityName Structural formula nmol/min (%) nmol/min (%)Methylαgalactopyranoside Galα-OMe 0.038 2.0 0.017 0.8Methylβgalactopyranoside Galβ-OMe 1.110 59.2 0.355 15.6Methylαglucopyranoside Glcα-OMe 0.008 0.4 0.007 0.3Methylβglucopyranoside Glcβ-OMe 0.009 0.5 0.005 0.2Methylαmannopyranoside Manα-OMe 0.010 0.5 0.005 0.2Methylβmannopyranoside Manβ-OMe 0.008 0.4 0.007 0.3N-Acetylgalactosamine GalNAc 0.228 12.2 0.050 2.2 N-AcetylglucosamineGlcNAc 0.008 0.4 0.007 0.3 Lactose Galβ1,4-Glc 1.875 100.0 2.277 100.0N-Acetyllactosamine Galβ1,4-GlcNAc 1.861 99.3 0.345 15.1 Methylβ-Galβ1,3-GlcNAcβ- 1.435 76.5 0.878 38.6 galactopyranosylβ1,3-N- OMeacetylglucosaminide

TABLE 4-2 Acceptor substrate specificity ofβ-galactoside-α2,6-sialyltransferase derived from strain ISH224-N1C0(No. 2) Acceptor substrate JT-ISH-224-N1C0 JT0160 SialyltransferaseSialyltransferase activity activity Relative Relative activity activityName Structural formula nmol/min (%) nmol/min (%)Methylαgalactopyranosyl Galα1,3-Galα-OMe 0.023 1.6 0.007 0.5α1,3-galactopyranoside Methylβgalactopyranosyl Galβ1,3-Galβ-OMe 0.96467.5 0.176 13.9 β1,3-galactopyranoside 2′-FucosyllactoseFuc-α1,2-Galβ1,4- 1.316 92.1 0.636 50.3 Glc Lactose Galβ1,4-Glc 1.428100.0 1.264 100.0

Example 7 Comparison of Acceptor Substrate Specificity TowardGlycoprotein Between JT-ISH-224-Derived Recombinantβ-Galactoside-α2,6-Sialyltransferase and Strain JT0160-Derivedβ-Galactoside-α2,6-Sialyltransferase (Known Enzyme)

As a glycosyl acceptor substrate, asialofetuin was used. Asialofetuin (2mg) was dissolved in 1 ml of 20 mM Bis-Tris buffer (pH 6.0) and used asa glycosyl acceptor substrate solution. As a glycosyl donor substrate,CMP-NeuAc was used. The glycosyl acceptor substrate solution (40 μl),the glycosyl donor substrate (5 μl) and either of the enzyme solutions(5 μl, 10 mU each) were mixed and incubated at 25° C. for 2 hours tocause sialic acid transfer reaction. After completion of the reaction,the reaction solution was gel-filtered by being applied to a SephadexG-50 Superfine (0.8×18.0 cm) equilibrated with 0.1 M sodium chloride. Aglycoprotein-containing eluate fraction (2-4 ml fraction) from gelfiltration was collected and measured for its radioactivity using aliquid scintillation counter to quantify sialic acid transferred to theglycosyl acceptor substrate.

As a result, both enzymes were found to have the ability to transfersialic acid to asialofetuin. Moreover, JT-ISH-224-derivedβ-galactoside-α2,6-sialyltransferase (N1C0) was found to achieve higherefficiency of sialic acid transfer thanβ-galactoside-α2,6-sialyltransferase derived from Photobacteriumdamselae strain JT0160.

TABLE 5 Acceptor substrate specificity ofβ-galactoside-α2,6-sialyltransferase derived from strain ISH224-N1C0(No. 3) Enzyme solution Radioactivity (cpm) JT-ISH-224-N1C0 3051 JT01602150 Absence 9

Reaction Conditions Reaction Composition:

Asialofetuin solution 10 μl  Enzyme solution 5 μl 5 mM CMP-sialic acid(in 20 mM cacodylate buffer 5 μl (pH 5)) + ¹⁴C-CMP-sialic acid

INDUSTRIAL APPLICABILITY

By providing a novel β-galactoside-α2,6-sialyltransferase and a nucleicacid encoding the same, the present invention provides a means forsynthesizing and producing sugar chains which are being shown to haveimportant functions in the body. In particular, sialic acid is oftenlocated at the nonreducing termini of complex carbohydrate sugar chainsin the body and is a very important sugar in terms of sugar chainfunctions. Thus, sialyltransferase is one of the most in demand enzymesamong glycosyltransferases. The novel sialyltransferase of the presentinvention can be used for the development of pharmaceuticals, functionalfoods and other products where sugar chains are applied.

1. An isolated protein comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, amino acid residues 15-514 of SEQID NO: 2, SEQ ID NO: 4 and SEQ ID NO:
 12. 2. An isolated protein havingβ-galactoside-α2,6-sialyltransferase activity, which comprises: (a) anamino acid sequence comprising deletion, substitution, insertion and/oraddition of one or more amino acids in an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, amino acid residues 15-514 ofSEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 12; or (b) an amino acidsequence sharing an amino acid identity of 60% or more with an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, aminoacid residues 15-514 of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO:
 12. 3.An isolated protein encoded by a nucleic acid comprising a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, nucleotides43-1545 of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO:
 11. 4. An isolatedprotein having β-galactoside-α2,6-sialyltransferase activity, which isencoded by a nucleic acid comprising: (a) a nucleotide sequencecomprising deletion, substitution, insertion and/or addition of one ormore nucleotides in a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, nucleotides 43-1545 of SEQ ID NO: 1, SEQ IDNO: 3 and SEQ ID NO: 11; (b) a nucleotide sequence sharing an identityof 70% or more with a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, nucleotides 43-1545 of SEQ ID NO: 1, SEQ IDNO: 3 and SEQ ID NO: 11; or (c) a nucleotide sequence hybridizable understringent conditions with the complementary strand of a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, nucleotides43-1545 of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO:
 11. 5. The isolatedprotein according to claim 1, which is derived from a microorganismbelonging to the genus Photobacterium.
 6. An isolated nucleic acidencoding a protein comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, amino acid residues 15-514 of SEQ IDNO: 2, SEQ ID NO: 4 and SEQ ID NO:
 12. 7. An isolated nucleic acidencoding a protein having β-galactoside-α2,6-sialyltransferase activity,wherein the protein comprises: (a) an amino acid sequence comprisingdeletion, substitution, insertion and/or addition of one or more aminoacids in an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, amino acid residues 15-514 of SEQ ID NO: 2, SEQ ID NO: 4and SEQ ID NO: 12; or (b) an amino acid sequence sharing an identity of60% or more with an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, amino acid residues 15-514 of SEQ ID NO: 2,SEQ ID NO: 4 and SEQ ID NO:
 12. 8. An isolated nucleic acid comprising anucleotide sequence selected from the group consisting of SEQ ID NO: 1,nucleotides 43-1545 of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO:
 11. 9.An isolated nucleic acid encoding a protein havingβ-galactoside-α2,6-sialyltransferase activity, wherein the nucleic acidcomprises: (a) a nucleotide sequence comprising deletion, substitution,insertion and/or addition of one or more nucleotides in a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, nucleotides43-1545 of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 11; (b) anucleotide sequence sharing an identity of 70% or more with a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, nucleotides43-1545 of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 11; or (c) anucleotide sequence hybridizable under stringent conditions with thecomplementary strand of a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, nucleotides 43-1545 of SEQ ID NO: 1, SEQ IDNO: 3 and SEQ ID NO:
 11. 10. An expression vector comprising the nucleicacid according to claim
 6. 11. A host cell transformed with theexpression vector according to claim
 10. 12. An isolated microorganismbelonging to the genus Photobacterium, which expresses the proteinaccording to claim
 1. 13. A method for producing a protein havingβ-galactoside-α2,6-sialyltransferase activity, which comprises thefollowing steps: 1) culturing a microorganism producing theβ-galactoside-α2,6-sialyltransferase according to claim 1; and 2)isolating the β-galactoside-α2,6-sialyltransferase from the culturedmicroorganism or the culture supernatant.
 14. The method according toclaim 13, wherein the microorganism producing theβ-galactoside-α2,6-sialyltransferase according to claim 1 isPhotobacterium sp. strain JT-ISH-224 (Accession No. NITE BP-87).
 15. Amethod for producing a recombinant protein havingβ-galactoside-α2,6-sialyltransferase activity, which comprises thefollowing steps: 1) transforming a host cell with an expression vectorcomprising the nucleic acid according to claim 6; 2) culturing theresulting transformant; and 3) isolating the protein havingβ-galactoside-α2,6-sialyltransferase activity from the culturedtransformant or its culture supernatant.
 16. An antibody, which binds tothe β-galactoside-α2,6-sialyltransferase protein according to claim 1.